210 DNA SYNTHESIS IN BACTERIA-FED Tetrahymena

34. Shortt, H. E. 1922. Review of the position of the genus Haemocystidium (Castellani and Willey, 1904), with a description of two new species. Indian J. Med. Res. 9, 814-26.

35. Wenyon, C. M. 1908. Report of travelling pathologist and

36. - 1926. Protozoology 2, Bailliere, Tindall and Cox, Lon- don.

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protozoologiut-3rd Rep. Wellcome Res. Lab. 12 1-68. 473-88.

J. PROTOZOOL. 14(2) , 210-213 (1967).

The Time of DNA Synthesis During the Interdivision Growth Cycle of Tetrahymena pyriformis Fed on Living Bacteria

JOHN CALKINS and GEORGE GUNN

Division of Radiobiology, Department of Radiology, University of Kentucky College of Medicine, Lexington, Kentucky

SYNOPSIS. It has been possible to obtain selective labeling of the macronucleus of Tetrahymena pyriformis fed on living Escherichia coli. The bacteria themselves, a thymidine requiring mutant, were labeled by exposure to tritiated thymidine in a lettuce infusion me- dium supplemented with trppticase broth. Various patterns of labeling

E DNA synthetic cycle of Tetrahymena pyriformis rIrHhas been reported only for axenic culture. The reported values for the macronuclear DNA synthetic period are indicated in Table 1.

The radiation response of a bacteria-fed strain of T . pyriformis isolated by one of us (J.C.) is being investi- gated in detail. It has been widely assumed that DNA and its synthetic period should be related to the response to radiation. I t is evident from the variety of values in Table 1 that the simple extrapolation of DNA synthetic period from one of the axenic culture determinations would be questionable. Therefore it seemed essential to locate the 3 periods related to DNA synthesis; i.e., GI, the pre-DNA synthetic period, S, the period of DNA synthesis and Gz, the period between the end of DNA synthesis and the next division, under the same culture conditions used in the radiation experiments. I t was expected that experi- ments involving 2 different living organisms would not produce as clear-cut results as those which can be obtained from a pure culture. This has proven to be the case; how- ever, it appears that the critical periods in the growth cycle can be determined even under the more complicated growth conditions.

MATERIALS AND METHODS

The general culture methods used have already been described(7) ; some modifications of culture methods have been required for label- ing experiments. Our strain of Tetrahymena, used in all experiments reported here, was isolated in Texas in 1960 and has been in bac- terized lettuce infusion medium since that time.

The general system of determining the DNA synthetic period followed the method of Berger and Kimball(8). In order to obtain nuclear labeling of the bacteria-fed animals, it has been necessary to feed the Tetrahymena on a n Escherichia coli mutant requiring thymidine; the mutant bacterial strain IT (S. S. Cohen) has been found to incorporate radioactive thymidine well enough to allow nuclear labeling of the Tetrahymena. Addition of radioactive thy- midine (a) to bacterial media without a period of baoterial growth, or (b) with non-thymidine requiring bacteria, has provided very poor labeling. Since good labeling was obtained only with bacteria

were Seen in synchron,ized Tetrahymena when the radioactive bacteria were given at panticular times during the growth cycle. These pat- terns have been interpreted as indicating the duration of the GI, S, and G, periods; they also suggest that a soluble pool of thymine exists in this animal from one S period to the next.

requiring ithymidine, and only after these bacteria had been per- mitted to grow in the radioactive media, it seems most likely that under experimental conditions the bacterial DNA was digested and used as the principal source of DNA precursors, a t least thymine, for the Tetrahymenu. We have no information on the exact cherni- cal products of bacterial digestion which Tetrahymena uses and which carry the radioactivity to the DNA; since the label is on the thymine, we use this term altho free thymine may not be a direct precursor of tetrahymenal DNA. A small addition of trypticase broth (1 ml per liter of lettuce medium) provided sufficient nutrients for good bacterial and protozoal growth. Therefore, this medium and bacterial strain IT- have been used in radiation experiments with Tetrahymenu as well as far the labeling experiments. To obtain radioactive bacteria, the day before an experiment E. coli IT- were inoculated into 2 flasks containing a small quantity of auto- daved medium (5 or 10 ml). To one flask of medium 4 +c per ml of P-thymidine was added; the second flask was identical except for the lack of radioaotive thymidine. The bacteria were allowed to grow at room tempemture for about 20 hours before the cultures were used in the experiments. The culture containing radioactive thymidine was designated as (‘hot” medium and the other L‘cold” medium.

Tetrahymenu usually grow well as isolated animals in 2-3 drops of medium contained in double depression slides; in the labeling experiments it was not uncommon to use 2-20 animals per depres- sion. There are sufficient nutrients in the 3 drops of medium for growth of 300-400 Tetrahymena; the animals in multiple culture grew in an identical manner to single animals.

Synchronized animals for the experiments were obtained by select- ing e d isd&ing animals in the visible stages of division from mass, log phase cultures; Figure 1 illustrates the typical degree of synchrony obtained by this method. Dividing animals were placed in drops filled with “hot” or “cold” medium according to the plan of the experiment. Free-hand pipetting under a low power stereoscopic micposcope was used for this and all subsequent manipulations of the animals. The aotual time from division to division was noted for different group of animals. It was found that the period varied somewhat within an experiment ; however, the average interdivision time shown in Figure 1 of about 2% hours was typical of all experiments.

By observing living aninnals, it was found that feeding was active thruout the growth cycle (1-2 minutes/vacuole). There seemed to be a reduction in the rate of food vacuole formation only about the middle of cytokinesis. Even before the 2 daughters separated very active feeding was resumed. The number of food vacuoles per

DNA SYNTHESIS IN BACTERIA-FED Tetruhyymena 211

animal (about 18) was somewhat reduced at time of division but it appeared that the excretion of vacuoles was depressed for the first 5-10 minutes after division and the active feeding quickly restored the average number. Since the number of food vacuoles per animal remained relatively constant t h o u t the early part of the growth cycle (at 18), and since formation and excretion occurred at rates of 1-2 minutes per vacuole, the complete digestion time would be no longer than 18-36 minutes. This time for vacuole processing agrees with the time to reach equilibrium in cytoplasmic radioactivity considered in the Results section.

Animals that were pipetted on gelatin “subbed” microscope slides for autoradiography died when the small drop of medium contain- ing them dried up. In the experiments r e p t e d here the animals to be autoradiographed were always placed on slides at the 1st or 2nd division following isolation; since it took a few minutes for the animals to dry, the dividing animal would occasionally separate into 2 small animals.

After the animals were allowed to dry thoroly on the slides, they were Feulgen-stained ; the acid hydrolysis is doubtlessly quite effective in removing any soluble thymine. After Feulgen staining the slides were dipped in Kodak NTB3 liquid emulsion to begin exposure; all exposure times for a given experiment were the same. The required exposures were relativly short, ranging from 45 min. to 2 hours; following exposure, slides were developed and fixed. Counts of the silver grains over the nucleus, cytoplasm, and an area equal to and adjacent to the animal (for background c m - tion) were made using a phase aontrast microscope. Bars in Figure 2 show typical statistical errors of grain count.

Two types of experiment were performed. In the 1st type, divid- ing animals were allowed to grow in “cold” medium for variable periods of time and were then transferred ko “hot” medium, where they were allowed to grow to division. They were then placed on slides for autoradiogmpby. The fraction of the cycle spent in “cold” medium w a s computed and plotted against the average number of nuclear and cytoplasmic grains. Figure 2 illustrates the 1st type of experiment.

The 2nd type of experiment, called “pulse labeling,” was similar to the 1st except that animals were not allowed to grow to the time

TABLE 1. Relative duration of the period of macronuclear DNA synthesis.

strain Macronuclear DNA synthesis Inveatigator Tetrahymena

W Throughout the cycle Walker & Mitchson(1)

H Begins some time after di- McDonald(2) vision and ends a con- siderable time before the next division

Mating Type I1 G, a few minutes to 2 hours

S probably similar in all cells 1-1s hours

Ga (including division time of about 20 min) 1%-2% hours

McDonald(3) var. I or more

HS S beginning at about 10% Prescott(4) and ending at about 50% of the cycle

and ending at about 67% of the cycle

HSM S beginning at about 2070 Stone & Prescott(5)

HSM Approx. 22% to approx. Cameroii & Stone(6) 68%

EU 6000 Approx. 19% to approx. 63 70

EU 6002 Approx. 16% to approx. 5570

TIME IN HOURS Fig. 1. Typical growth of animals synchronized by physical selec-

tion and isolation of dividing animals from log phase cultures. Only the first cycles were used for experiments. The growth cycle is considered to start at the time animals are selected (during division).

of division in the crhot” medium but were removed following a 20 minute labeling period. A sample of the animals was dried on the slides at the time of the 1st division, while mather sample of the animals was bransferred to a depression containing cold medium and allowed to grow until the 2nd division occurred. At this time they were then dried on the slides. Figure 3 illustrates the general plan of the 2nd type of experiment.

RESULTS AND DISCUSSION

Figure 2 is a plot of the average number of nuclear and cytoplasmic grains of animals placed in radioactive medium at different times thruout the division cycle and allowed to grow to the next division. Each point in this figure was computed from counts on 15-20 animals. The standard errors of the grain counts indicated by the Bars are rather high. I t was observed, in both living animals (using phase contrast microscopy) and in the animal stained for auto- radiography, that the macronucleus is a relatively flexible structure in which the food vacuoles can become deeply embedded. When food vacuoles are unfavorably located in an animal fixed for autoradiography, they can effectively shield much of the macronucleus from the autoradiography emulsion. This factor doubtlessly contributed to the large variation in grain counts from individual to individual and thus produced the large individual standard errors noted. With the close spacing of points in Fig. 2 the trend of the labeling pattern seems clear ; this experiment has been re- peated several times with essentially the same result. There seem to be 3 phases of the growth cycle indicated by the level of nuclear labeling. Referring to Fig. 2, there is a period during which the nucleus is relatively heavily and uniformly labeled. Altho there is some variability in the experimental results, this appears to be the first 20% of the growth cycle. This period is interpreted to be the GI period.

Following this (GI) period there is a period of progres- sively decreasing nuclear labeling. The later the animals

2 1 2 DNA SYNTHESIS IN BACTERIA-FED Tetrahymena

I I I I I F U I

P E R C M OF CVCLE COYRETED o m 2 0 a o 1 0 5 o w ~

Fig. 2 . .4verage number of nuclear ( X ) and cytoplasmic (0 ) grains observed in animals transferred to radioaotive medium after growth in cold medium for various times. Points are plotted at the percent of cycle completed before transfer. Lines are fitted <‘by eye.’’ and approximate durations of GI, S and G2 indicated by this experi- ment are shown.

were put into the radioactive medium during this period, the less radioactivity was observed in the nucleus. These observations suggest that DNA synthesis is occurring dur- ing this period; it would therefore correspond to the S period. The end of the period is somewhat uncertain, but is appears to be a t about 60% of the way thru the growth cycle. Following the S period, (about 60% of the cycle completed) and lasting to the next division, the nuclear grain counts were not significantly different from back- ground grain counts, as would be expected for the G2 period.

The fractions of the interdivision growth cycle occupied by G1, S, and Gz appear to be quite similar to the fractions shown by Stone & Prescott(5) and Cameron & Stone(6), who indicated nuclear labeling from about 20-30% to 55- 70% of the growth cycle. Stone, Miller, and Prescott(9) noted that the beginning of S, as determined by tritiated thymidine given during a given growth cycle, could be somewhat in error because of a possible (unlabeled) nu- clear pool of soluble thymidine derivatives which animals might use to initiate DNA synthesis before transporting exogenous (labeled) thymidine into the nucleus for DNA synthesis. The S period, in our case, may be somewhat longer than indicated by the nuclear labeling because of possible delay between the time of ingestion of radioactive bacteria and the availability of radioactive thymine for DNA synthesis; this delay must be rather short or the pulse label in GI (Fig. 3) would produce heavy labeling when the S period begins, the animals having been removed from the radioactive bacteria less than 15 minutes before the start of the S period. I t is rather interesting to note that the animals appear not to grow progressively more radioactive with increasing duration of exposure during GI (Fig. 2 ) . This result has been observed uniformly in several experiments and would seem to imply that Tetra- hymenu does not stockpile the radioactive thymine ob- tained from the bacteria ingested during G1 either as DNA

or as soluble thymine. Fig. 2 and the pulse labeling ex- periment to be described suggest that the Tetrahymena are excreting bacterial DNA or its metabolic product, with very little delay except during the S period. Noting (Fig. 2 ) the level of cytoplasmic labeling (mostly associated with food vacuoles), we find that the cytoplasm quickly comes to equilibrium with progressive exposure to radio- active bacteria; i.e., animals exposed for more than 20% of the cycle (because of the design of the experiment the non-equilibrium appears a t the end of the cycle) were eliminating radioactivity as fast as they were gaining it by ingestion. The cytoplasmic grains indicate only the in- soluble forms of thymine and its products, presumably bacterial DNA, which the animal contains. The equilibrium noted above could occur by digestion of bacterial DNA (rendering it soluble with subsequent removal by staining procedure) ; however, since no stockpiling of thymine dur- ing G1 is evident, it seems more likely that bacterial DNA is simply excreted undigested except during the S period.

The results of the pulse labeling experiment plotted in Fig. 3 tend to substantiate the interpretation of the events shown in Fig. 2. It was anticipated that all animals in the 2nd type experiment wauld show some level of nuclear labeling. The animals placed in the radioactive medium during the G1 period would probably have some bacteria in an undigested form in the cytoplasm at the beginning of the S period which would provide radioactive thymine as they were digested during S. Animals placed in radio- active medium during the S period would be expected to show the most nuclear labeling and, since there is some straggling in progression thruout the cycle, groups of animals placed in radioactive medium during the early G2 period might still contain individuals that had not completed the entire S period and thus show nuclear label- ing. As can be seen in Fig. 3, all groups of animals do show some nuclear labeling. Since the pulse labeling period occu-

FIRST CVCLE SECOND CVCLE PERCENT OF THE INTERWISION GROWTH CYCLE COMPLETED

Fig. 3. Plan of the pulse labeling experiment (shown at the top) as related to the labeling shown in Fig. 2 (lower left). The bar graph (lower right) shows average nuclear (solid) and cytoplasmic (open) grains for the different groups. Arrows on groups 2 , 4, and 6 show grain counts (% values for groups 1, 3, and 5 respectively) expected if all tritiated thymine present at the first division were incorporated in DNA (no soluble pool).

DNA SYNTHESIS IN BACTERIA-FED Tetrahymena 2 13

pies more than the total G1 period, and yet the nuclear grains of GI animals are only about those of animals ex- posed to radioactive medium during S, animals exposed to radioactive bacteria early in GI must be able to excrete rapidly the radioactivity from the DNA ingested during early GI.

Arrows on the bar graph in Fig. 3 indicate the number of grains expected in animals dried after the 2nd cycle on the assumption that the labeled protozoal DNA was divided between 2 daughter cells. I t will be noted that the level of labeling after a 2nd cycle in cold medium is in all cases somewhat above the expected amount. I t is difficult to say whether the increase above the expected amount is statistically significant since the absolute grain counts de- pend on rather erratic factors such as how well the animals flatten when dried on the slides for autoradiography. Ani- mals that flatten into a very thin layer will produce higher nucleus during the S period the more likely it would be to go into the soluble pool and the less likely would be its because of the low penetrating power of the tritium beta particles. The results in Fig. 3, however, strongly suggest the presence of a soluble pool of thymine persisting from one S period to the next; probably in the nucleus as shown by Stone, Miller, and Prescott(9). If a pool persisted it would be expected that the later the thymine reached the nucleus during the S period the more likely it would be to go into the soluble pool and the less likely would be its incorporation in the DNA being synthesized. If the radio- active thymine taken in during GI can be interpreted as being available early during the S period, the S pulse label supplying thymine about the middle of the S period, and the pulse labeling during GZ providing thymine for the late labeling of stragglers which have not yet completed the S period, then it would be expected that the excess nuclear grains following division would be progressively more pronounced from GI, to S, to GP in accord with the ob- servations (Fig. 3).

Comparing cytoplasmic grain counts shown in Figs. 2 and 3, it is seen that the growth period in cold medium has eliminated a large part of the cytoplasmic grains. The cytoplasmic labeling from the S period pulse (Fig. 3) should have been less than that from the Gz pulse if it were simply residual bacterial DNA (group 3 vs. 5 and 4 vs. 6) ; the fact that the cytoplasmic grains from the S

period pulse exceed those from the G2 suggests a more com- plex relation. It should be noted that cytoplasmic incorpo- ration of H3-thymidine has been found even in the cultures in which only thymidine and not bacterial DNA was in- gested by the animals(3,5,10,11). Altho mitochondria1 DNA has been found in T . pyriformis ( 1 1) , the cytoplasmic synthetic period in our animals does not seem to agree with that found in other systems( 11).

In summary, it seems that autoradiography can supply information about biochemical aspects of the growth cycle of Tetrahymena even when the labeled precursors are administered via another living organism. The results we obtained seem to be quite similar to the recent findings of other investigators using different strains of this organism and feeding thymidine directly to the Tetrahymena.

We should like to acknowledge the assistance of Yvonne Gover, Teddie Hamman, and Doris Gaines in the experiments reported here. This research was supported by Grant GM 12030-02 from the Insti- tute of General Medical Sciences of the NIH.

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2. McDonald, B. B. 1958. Quantitative aspects of deoxyribose nucleic acid (DNA) metabolism in an amicronucleate strain of Tetra- hymena. Biol. Bull. 114, 71-94.

3. - 1962. Synthesis of deoxyribonudeic add by micro- and macronuclei of Tetrahymena pyriformis. J . Cell Biol. 13, 193.

4. Prescott, D. M. 1960. Relation between cell growth and cell division. IV. The synthesis of DNA, RNA, and protein from division to division in Tetrahymena. Exp. Cell Res. 19, 228.

5. Stone, G. E. & Prescott, D. M. 1%4. Cell division and DNA synthesis in Tetrahymena pyriformis deprived of essential amino acids. J . Cell Biol. 21, 275.

6. Cameron, I. L. & Stone, G. E. 1964. Relation between the amount of DNA per cell and the duration of DNA synthesis in three strains of Tetrahymena pyriformis. Exp. Cell Res. 36, 510-4.

7. Calkins, J. 1964. The lethal effects of radiation on six species of Protozoa. Photochem. Photobiol. 3, 143.

8. Berger, J. D. & Kimball, R. F. 1964. Specific incorporation of precursors into DNA by feeding labeled bacteria to Paramecium aureliu. J . Protoeool. 11, 534.

9. Stone, G. E., Miller, 0. L. & Prescott, D. M. 1965. Ha-thymidine derivative pools in relation to macronuclear DNA synthesis in Tetrahymena pyriformis. J . Cell Biol. 25, 171.

10. Scherbaum, 0. H. 1960. Possible sites of metabolic control during the induction of synchronous cell division. Ann. N. Y . Acad. Sci. 90, 565.

11. Cameron, I. L. 1966. A periodicity of tritiated-thymidine incorporation into cytoplasmic deoxyribonudeic acid during the cell cycle of Tetrahymena pyriformis. Nature 209, 630-1.

IN MEMORIAM

RICHARD R. KUDO

25 July 1886 - 3 June 1967