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Studies on the mechanism of serotypic transformation in Paramecium aurelia
0 1967 by heademic Press Experimental
Inc.
Cell Research 45, 289-305 (1967)
STUDIES
ON THE MECHANISM
FORMATION I.
THE
EFFECTS
OF
ACTINOMYCIN
INDUCED
L.
AUSTIN,
OF SEROTYPIC
IN PARAMEC1UM
CHLORAMPHENICOL
MARY
289
ON
D, AN
TRANS-
AURELIA PUROMYCIN,
AND
ANTISERUM-
TRANSFORMATION’
J. PASTERNAKs
and BERTINA
Department of Zoology, Indiana University, Bloomington, Received
M. RUDMAN Ind., U.S.A.
June 13, 1966s
THE basic features of the surface antigen system of Paramecium aurelia have been described and discussed in numerous papers, e.g., in those of Sonneborn [38, 39, 411, of Beale [B-9], of Preer [30, 311, of Beale and Wilkinson [la], of Kimball [23], and of Allen [a]. Briefly, each genitally homozygous clone of P. aurelia has a repertory of serotypes (antigenic types, immobilization antigens, or surface antigens); in stock 51, syngen 4, for example, there are at least 12 non-allelic loci, each of which determines a specific antigenic type. A distinguishing feature of the serotype system in P. aurelia is that at one time in any cell only one antigenic type determined by a single gene locus is expressed despite each cell’s potential ability to determine an array of different antigenic types. Only a few exceptions to this rule of mutual exclusion have been reported [16, 241. Which particular antigenic type is expressed is not merely gene-dependent but is also influenced by the state of the cytoplasm (prior and prcscnt), by the stage in the life cycle (whether autogamy-immature or autogamy-mature) [ 141, and by various extrinsic environmental factors, such as temperature, type of culture medium, pH, salt content of the medium, homologous antiserum, radiation, enzymes, and the antibiotic patulin [3] (see reviews listed above). The ciliary antigens have been found to coat the cell surface as well as the cilia
[lo,
11, 15, 34, 451. They are known to be fibrous
heteropolymeric
r Contribution no. 793 from the Department of Zoology, Indiana University. This investigation was supported mainly by Public Health Service Research Grant no. 5-KOl-Ca 05398-03-05 from the National Cancer Institute (to M. L. A.), and in part by Genetics Training Grant PHS-GM-82-05-06 (to J. P.); and was aided by contract COO-235-21a from the Atomic Energy Commission and grant no. E-81H from the American Cancer Society to T. M. Sonneborn. a Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada. 3 Hevised version received October 10. 1966. Experimental
Cell Research 45
Mary I,. Austin, J. Pasternak
and Bertina
M. Rudman
polypeptide structures [al, 32, 441, and within a stock the different antigenic types determined by non-allelic loci have been found to possess divergent immunological and physico-chemical properties [ 13, 33, 421. Moreover, the amino acid content of the different antigens varies significantly [al, 22, 43, 441, an observation which suggests that a change from one antigenic type to another within a clone probably does not entail refolding of the existing protein structure, but more likely involves de novo protein synthesis of all or part of the “new” antigen [4, 21, 431. Sonneborn [38] was the first to demonstrate that exposure to homologous antiserum (AS) stimulated transformation, that the direction of transformation (i.e., the serotype to which the animals were transformed) could be controlled by prescribed temperatures and/or the quantity of food supplied daily, that transformations were reversible, and that certain diverse serotypes could be maintained during reproduction more or less indefinitely under identical conditions of culture. The present investigation was undertaken to enlarge our understanding of the nature of the biosynthetic processes during transformation, and to gain some insight into the mechanisms that maintain a particular antigenic type. To test the hypothesis that these processes and mechanisms inc,lude gene-controlled synthesis of the antigenic proteins, the effects of three antibiotics, actinomycin D (,4MD), puromycin (PURO), and chloramphenicol (CM), were investigated. The actions of these antibiotics are reasonably well known [1, 17-19, 35, 36, 46, 471. All three antibiotics were found to affect the transformation of serotypes; the particular one studied here was the antiserum-induced transformation of 5lD to 51R. (The different serotypes in P. aurelia are designated by capital letters, the different stocks by numbers. Since only stock 51 was used in this study, the serotypes will hereafter usually be referred to by the letter alone.) As will appear, the effects of the antibiotics vary in dependence upon their concentration, the prevailing temperature, the concentration of the homologous antiserum used to induce transformation, and the time relations between exposure to homologous antiserum and exposure to antibiotic. The interrelations among these variables provide insight into the nature of the process of serotype transformation. The most remarkable and informative aspect of the results is that each antibiotic either inhibits or stimulates serotype transformation in definite and regular dependence upon the particular combination of variables.
Experimental
Cell Research 45
Effects of antibiotics
MATERIALS
on transformation
AND
in P. aurelia. I
291
METHODS
Growth of cultures prior to experiments; selection of paramecia for testing.-Cultures were prepared for the experiments as follows. Single washed cells of P. aurelia, serotype D of stock 51, syngen 4, with genotype KK, lacking kappa, were grown up into tube cultures and maintained in 0.15 per cent cerophyl, containing 0.6-0.9 g 12H,O)/l. This medium was used 24 hr of dibasic sodium phosphate (Na,HPO,. after inoculation with Aerobacter aerogenes at which time the pH was 7.0-7.2. Enough inoculated cerophyl medium was added daily to the cultures to maintain reproduction at the rate of one fission per day [29,40]. When used for an experiment, such cultures might have passed through as many as three autogamous periods since the initial isolation, but, if selected for an experiment, a culture had at that time no more than 5 per cent of the animals in autogamy. At the start of an experiment, always 24 hr after the last feeding, the culture was usually>97 per cent pure for serotype D. Chemicals.-Puromycin dihydrochloride was purchased from Nutritional Biochemicals Corporation; chloramphenicol from Parke, Davis and Company. We acknowledge with thanks the gift of actinomycin D from Merck, Sharp and Dohme, West Point, Pa. Procedure.-Experiments were carried out as follows. From the preparatory tube cultures four aliquots of about 3000 cells in 10 or 12 ml of culture medium were put into 50 ml flasks. The culture medium and pH used for the experiments were those which in general favour the appearance and long maintenance of 51B, i.e., baked lettuce medium adjusted to a pH of about 6.8 by the use of sodium phosphate buffer. To one flask was added an antibiotic, to the second homologous antiserum, to the third both, and to the fourth neither. All flasks were brought to the same final volume, for example, by adding distilled water instead of antibiotic, or non-homologous antiserum instead of homologous antiserum. Since cultures of D at times had up to 3 per cent contamination with serotypes A, C, and/or E, antisera against these types were added along with the anti-D serum in the same non-lethal concentration as was the anti-D serum (given below). At intervals after the flasks were prepared, samples were withdrawn and tested for serotype. Animals were scored as being of a certain serotype only when they were completely immobilized by that particular antiserum [3]. Experiments were run in varying concentrations of the AS (1: 2400 up to 1: 800, none of them lethal) as well as of the antibiotics. Most of the different combinations of concentrations of AS and of antibiotic (high AS with high antibiotic, low with low, and reciprocal combinations) have been tested at 27°C with certain ones also tested at 14”, 19”, and 31°C. In general, each experiment of a kind has been run at least twice, and some repeatedly. Details will be given as the experiments are reported under Results. Inhibition and stimulation were detected operationally by comparing the percentages of transformed cells in controls lacking antibiotics with the percentage in experimentals exposed to antibiotics. Such comparisons, made at intervals during the course of an experiment, made it possible to detect effects on speed of transformation of a culture up to the maximum percentage transformed. Such effects do Experimental Cell Research 45
292
Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
not necessarily imply an effect on the speed of transformation of individual cells because the moment of initiation of the transformation processes could not be directly ascertained, and delays or accelerations in the final completed transformation of the cells (the observed phenomenon) could have been due to variations in the onset of the initiation of their transformation. Thus inhibition and stimulation of transformation of a culture took three observable forms: (1) later or earlier onset, and/or (2) retardation (deceleration) or hastening (acceleration) of transformation, and/or (3) reduction or increase in the final percentages of transformation as compared with those in the AS controls. Hence it is important to define the end point of the experiments. In general, experiments were terminated when deaths began to appear in one or more cultures or when the percentage of transformation plateaued in the controls exposed to antiserum alone, i.e., by 48 hr. Usually deaths did not occur within 48 hr and the antiserum controls exhibited maximal transformation by that time or sooner.
RESULTS Effects on the cells, other than on antigen alone and of antiserum (AS) alone
transformation,
of the antibiotics
Higher concentrations of the three antibiotics had deleterious effects on the paramecia, but lower concentrations did not. Little or no effect on fission rate was produced by concentrations of up to 1 ,ug/ml of AMD, up to 10 ,ug/ml of PURO, and up to 40 pg/ml of CM. However, division was suppressed without occurrence of deaths during three days of exposure to 2-3 pg/ml of AMD, to 25-80 pg/ml of PURO, and to 50 ,ug/ml-2.0 mg/ml of CM. Certain abnormalities in the paramecia were noted in concentrations equal to or greater than 5 rug/ml of AMD (the macronuclei tended to be enlarged or broken into 2-4 pieces), 50 ,ug/ml of PURO (the shape of some of the animals tended to be abnormal), and 500 ,ug/ml of CM (the animals tended to be large and dark). Deaths occurred on the third day in concentrations of 5-15 ,ug/ml of AMD and as early as the second day in 25 pg/ml. In PURO animals died on the third day in 100 ,ug/ml; no tests were carried in concentrations stronger than this. The strongest concentration of CM used was 2.0 mg/ml in which, as noted, no dying occurred. Antisera were used in concentrations of 1 : 800, 1 : 900, 1: 1200, and 1 : 2000 repeatedly. In addition, the concentrations of 1: 1600 (in three experiments), of 1: 1800 (in one experiment), and of 1: 2400 (in two experiments) have been tested. Except for a progressive reduction in mobility as the concentration increased, no deleterious effects on the paramecia were produced by any of these concentrations of antiserum. Experimental
Cell Research 45
Effects of antibiotics Eflects on antigen one temperature
transformation
on transformation of varying
in P. aurelia. I
293
the serum concentration
alone at
At each of the four temperatures (14”, 19”, 27”, and 31°C) increasing the serum concentration alone results in general in a progressive increase in the rapidity with which the percentages of transformed cells increase in a culture. This fact is made clear by the following mean percentages of transformation in different concentrations of antiserum at 19°C. After 9 hr of exposure in antisera 1: 2400, 1: 2000, 1: 1600, 1: 1200, and 1: 800, the mean percentages of transformation in this order were 0, 5, 5, 77.5, and 89.5. The maximum percentages of transformation obtained in these concentrations when the experiments ended at 48 hr were, in the same order, 45, 72 (at 24 hr), 78, 100, 98. Similarly, increasing percentages of transformed paramecia were obtained for antiserum concentrations of 1 : 2000, 1 : 1200, and 1 : 800 at both 27” and 31°C. Although the differences described can be slight in adjacent concentrations, Table I shows that they are quite marked in the more widely separated concentrations of 1: 2000 and 1: 800. Effects on antigen transformation antiserum concentration
of varying
the temperature
alone at one
The speed of transformation is also increased by increasing the temperature while keeping the antiserum concentration the same. The temperature effect is clearly shown by comparing the 6-hr results for the 1: 800 concentration (0, 40.5, and 100 per cent transformed) and the 8-hr results for the 1: 2000 concentration (2, 22, and 85 per cent transformed) at the three temperatures used (Table I). The relative effects of temperature transformation
and of antiserum
concentration
on speed of
When the relative effects of temperature and of antiserum concentration on speed of transformation are compared (Table I), it is clear that the temperature effect predominates, since at 31” the paramecia in almost the lowest concentration used (1: 2000) transform at a faster rate than they do in the highest concentration at either of the other two temperatures. This strong influence of temperature on the speed of transformation cannot be attributed to a general tendency of B to emerge at high temperatures, because in stock 51 serotype B is predominantly a low-temperature type [38] and because D control cells without antiserum, under the conditions and duration of these 19 - 671812
Experimental
Cell Research 45
Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
experiments, never transformed to B on exposure to increased temperature alone. Thus the temperature must act, not in directing the transformation to B, but in speeding up this transformation. Presumably this speeding up is accomplished through the general effect of temperature on rates of reactions. Acquiring control of the speed of transformation in the ways described by adjusting antiserum concentration and temperature has proved to be of importance in the experiments with the three antibiotics, AMD, PURO, and CM. Effects of AMD on antigen
transformation
Table II shows in summary form the occurrence and general extent of inhibition or stimulation of the D-to-B transformation produced by combinations of AS with 0.5 ,ug/ml of AMD. As we have seen, an antiserum concentraTABLE
I. Speed of transformation of serotype D to B at different and in different concentrations of antiserum.
temperatures
This is a composite table with each value representing the mean of transformation obtained in all experiments for a particular antiserum concentration, temperature of observation. The number of experiments used in calculating each mean is shown in following the percentage figure; the maximum number shown in a parenthesis in represents the total number of experiments run with that combination of antiserum tion and temperature. As is evident, observations were not made in all experiments Time of scoring tests W) 3 4 5 6 7 8 9 10 12 14 16
Concentrations At 19°C --1:soo
Experimental
1:800
At 31°C 1:2000
r--7 1:soo
1:2000
60 (2) 0 (2) 0 (3) 64 (2) 89.5 (1) 97.5 (1) 93 (3) 100 (2)
0 (2) 0.5 (2)
2 (2) 5 (2) 13 (3) 28 (3)
0 (5) 14 (9) 40.5 (9) 76.5 (9)
0 (2) 0 (2) 8 (13) 18.5 (11)
87 (3) 94 (3) 100 (3) 99.5 (3)
11 (2) 54 (2) 81 (2) 82 (2)
93 (6) 97 (8) 99 (‘3) 99 (6)
22 (10) 50 (11) 52.5 (13) 66 (13)
100 (3)
85 (2)
66 (2)
-
78.5 (2)
65 (3) 89.5 (5) 96.5 (3)
18 20 24 48
of 51D-antiserum At 27°C
1:2000
percentages and time parentheses any column concentraat all hours.
98 (3) 98 (3)
72 (3)
Cell Research 45
100 (7)
79.5 (9) 88 (3)
97.5 (3)
96
(2)
93
(2)
Effects of antibiotics on transformalion
295
in P. aurelia. Z
tion of I : 800 and a temperature of 31°C both hasten the onset of transformation. When AbfD (0.5 ,q/ml) is used simultaneously with these factors that cause rapid transformation, inhibition always results. When a combination of conditions are employed which now slow down ~ransformt~on in the AS controls, a progressively greater stimulatory effect results on the combinations of AMD and serum. When this concentration of AMD is used in combination with 1: 1200 AS at 27”C, tral~sformation appears to be usualIS inhibited, hut sometimes stimulated; the conditions here are apparently ~~~~~~~~ o( e#j%Trs oic‘ ~~~~t~~~~~~ D (0.5 ~~~~~~ irr ColttbiRatiorl with temperctture in inhibiting UPstimulating transformation of D to B induced by difrerent concentrations of antiserum (AS).
TABLE If.
The numbers of readings falling within 3 classes of percentages of inbibitition or stimulation (O-10%, 11X1-50%, and 50,1-100X) during transformation are shown. The fraction represents those experimentsof the total number In which the particular effect (inhibition or stimulation) was found under each set of conditions.
O-10 10-50 50-100
31°C x-w O--l0 10-50 50-100 O-10 IO-50 50-100
96Classes.. ,
a-10 IO-50 50-100 7
f:SOO
13
0 3 3 ‘Inhibition In 2/2
1 5 8 Inhibition In 3/3
27°C.
19°C
14OC 1
AS cont.
0
?Ximulat.ion in 7/7 expts
0 5 7 Inhibition in 212
1:soo
a 8 14 Stimulation in 6/6 expls
No test
2 7 0 Inhibition in 2/Z
1:12oa
a 4 a Stimuiation in 212 expts
3 3 0 StimuIatian in 212
0 2 (stim.) 0 a 5 Inhibitjon in 2.513
0 8 4 Inhibition in 212
1:2aoo
No test
6 0 1 Stimufation in Z/2
*5 10 0 Stimulation in 4j4
4 5 0 Inhibition in 2p
Differences between duplicate AS-controIs’
7
0
0
7 readings in 4 expts
16
1
0
17 readings in 4 expts
62
5
0
67 readings in 14 expts
hro test
45
2
0
47 readings in 10 expts
a See paragraph on Procedure for three ways in which stimulation or inhibition may be manifested. * C&e experiment in 1: 1800 antiserum inehtded here with three in 3 :2000 antiserum. ’ C)uplicate antiserum tests of the D-to-B transformation have been carried in a total of 32 experiments run at different times, with a totar of 138 readings made during transformation. Different ~on~e~tra~~onsof AS have not been separated in this summary, since there was no correlation between the concentration and the degree of difference in transformation percentages of the paired AS-controls.
296
Mary L. Austin, J. Pasternak
and Bertha
M. Rudman
borderline. However, when it has been used in experiments with 1 : 1200 AS at 19” and 14°C and with 1: 2000 at both 19” and 27”C, transformation has always been stimulated. In other words, the antibiotic stimulates or inhibits the transformation brought about by antisermn, depending upon the conditions employed in the experiment. When the consistency of these relations of temperature and AS concentration became apparent, it was predicted that stimulation with 1: 800 AS might be obtained at a temperature lower than 19°C. When this high concentration of AS was tested at 14°C with 0.5 pg/mI of AMD, stim~~lation did, in fact, result, as Table II shows. Table III shows specific results of some experiments in which transformaTABLE III.
Experimerit
la.a 1.
Inhibitory effect of acti~lomyci~ D (A~~~) on the tra~s~orI~ation of 5lD to 51 B in 1:800 antiserum (AS) at 19°C. Addition of AMD (hr) relative to that of AS
-3 0 +3 10
Presence of AS and concentrations of AMD (~g/ml)
Percentages of transformation to 518
Control ( -AS, - AMD) AS AMD (12.5) AMD (12.5) -I-AS AMD (12.5) i AS AS s AMD (12.5) AS -i-A&ID (12.5)
20 hr 0 95 0 0 0 2 7x
48 hr 0 97 0 Dead 14 40 97
0
Control ( -AS, -ARID) AS AMD (5) AMD (5) -I-AS AMD (1) AMD (1) -t-AS
13 hr 0 98 0 0 0 2
24 hr 0 98 0 1.5 0 18
48 hr 0 98 0 9 0 60b
0
Control ( -AS, - AMD) AS AMD (0.5) AMD (0.5) + AS
14 hr 0 100 0 0
24 hr 0 100 0 0
48 hr 0 96.5 0 67’
2.
0
3.
a Expt. la run at different time from Expt. 1; controls of la behaved like those of 1. ’ Includes 16.5 % of types other than B. ’ Includes 41 X of types other than Il. Experimental
Cell Research 45
Effects of antibiotics
on transformation
297
in P. aurelia. I
tion was inhibited. The antiserum concentration was 1: 800, the temperature 19”C, and the AMD concentrations employed were 12.5, 5, 1, and 0.5 ,ug/ml. When 12.5 ,ug/ml (Table III-l) was added simultaneously with, 3 hr before, and 3 and 9 hr after the antiserum, AMD proved to be a potent inhibitor, although, as the table shows, the later it was added to the antiserum, the less inhibition of transformation resulted. Similarly a concentration of 5 ,ug/ml of AMD (Table 111-2) gave a drastic inhibition of transformation through 48 hr. The lower concentrations of 1 ,ug/ml (Table 111-2) and of 0.5 ,ug/ml (Table 111-3) also reduced the amount of transformation significantly through the first day, but in the second 24 hr animals exposed to these concentrations transformed substantially, although they failed to attain the control level by the end of 48 hr of observation. TUI.E
IV.
Combinations of antiserum (AS) and actinomycin D (AMD) that accelerate the AS-induced transformation of 5lD to 51 B. Part A shows effects of using a high concentration of AMD with a very low concentration of AS from 0 time; part B shows effects of using a low concentration of AMD 3 hr before a high concentration of AS. Compare combinations that accelerate with those that inhibit transformation.
Addition of AMD (hr) relative to AS
Percentages of transformation to 51B after antiserum
Concentrations of AMD @g/ml) and of AS
24 hr Expt
(-4) 19°C 0 0 0
Control ( -AS, -AM A4MD (12.5) AS (1: 800) AS (1: 800) + AMD AS (1: 1600) AS (1:1600)+AMD AS (1: 2400) AS (1:2400)+AMD
D)
1
Expt
0 0 95 0 64 44 14 26
2
0 0 100 0 56 0 20 88 8 hr
Expt
(B) 19°C -3 0 +3
Control ( -AS, AMD (0.5) AS (1: 800) AMD i-AS AMD+AS AS+AMD
- AMD)
1
Expt
0 0 65 93 0 Experimental
2
0 0 78 0 0 Cell Research 45
Mary L. Austin, J. Pasternak
298
and Bertina
M. Rudman
Table IV shows some examples of stimulation of transformation at 19”C, in contrast with inhibition in the same experiments, when AMD was used in two widely separated concentrations. Table IV-A shows the results of two experiments with a high concentration of AMD, 12.5,~g/ml. These demonstrate that, although this high concentration drastically inhibits the transformation produced by 1: 800 antiserum and less drastically inhibits that brought about by 1: 1600 antiserum, it can stimulate transformation when the initial transformation-inducing stimulus is very weak, as it is in 1: 2400 antiserum; in other words, when the progress of transformation in the controls is quite slow. Table IV-B shows that stimulation of transformation can also be obtained when 0.5 ,ug/ml of AMD is given 3 hr prior to exposure to 1:800 AS at 19°C. We have previously pointed out that a third method of slowing down transformation in the controls and of obtaining stimulation is possible, i.e., by lowering the temperature (Table II).
4
5
6
7
8
9
IO
II
12 HOURS
AFTER
ANTISERUM
Fig. l.-The effects of actinomycin D on antiserum-induced transformation in P. aurelia at 27°C. (A) This figure shows the effects of 0.5 ,ug/ml of actinomycin D on the 51 D-to-51B transformation induced with two concentrations of homologous antiserum in one experiment. Transformation percentages in cells treated with homologous antiserum alone are shown for concentration 1: 800 as 0-0, for 1:2000 as A-A; with actinomycin D alone, as W-W; with culture fluid alone, as 0-n; and with actinomycin D in combination with 1:800 antiserum, as O-O, with 1:2000 antiserum as A-A. (B) This figure shows the results of a duplicate experiment. The experimental conditions and the symbols used are the same as those described under A. Experimental
Cell Research 45
Effects of anfibiofics
on transformation
in P. aurelia. I
299
Figs. 1 A and 1 B show graphically the results of two experiments in which both inhibition and stimulation of transformation were obtained by the same concentration of 0.5 pg/ml of AMD, this time at 27°C and combined with 1:SOO and with 1: 2000 antiserum. The usual controls were run in flasks containing culture fluid alone, AMD alone, and each of the antiserum concentrations alone. With the faster transformation, obtained by the use of antiserum 1:800, AMD inhibited transformation, vvhereas the same concentration of AMD stimulated transformation of the more slowly transforming group in antiserum 1: 2000. It should be noted that AMD treatment alone seldom caused serotypic changes in the first 48 hr. Out of the total of 50 experiments run to test the effect of AMD (with concentrations ranging from 0.125 pg/ml to 25 pg/ml) in only two instances, both from the same experiment, was there any transformation in AMD alone before the end of 48 hr. In these cases, D cells transformed after 30 hr 26.5 per cent to B and 70 per cent to N when treated with 3 pg/ml of ,4MD and 100 per cent to N when treated with 6 pg/ml of AMD. In general, such transformation occurred only on the third day and only in the lower concentrations (2-6 pg/ml), if it occurred at all. It should also be noted that in four experiments low concentrations of AMD (Q 5 pg/ml) used in conjunction with non-homologous antiserum never stimulated transformation above that in the AS control. Efecfs of PURO and CM on antigen
transformation
Similarly designed experiments were performed to ascertain the effects of PURO and of CM on antiserum-induced antigenic transformation. PURO (100 ,ug/ml) and CM (2 0.5 pg/ml) inhibit the transformation of D to B in 1: 1200 concentration of homologous antiserum. Lower concentrations of PURO (2.5 pg/ml) and of CM (40 pg/ml) combined with antiserum concentrations of 1: 800 and 1: 1200 stimulate the transformation, the stimulation being greater in the latter concentration than in the former. The results for the 1: 1200 concentration are shown in Table V. Thus it is clear that, even though the three antibiotics act at different sites in the cell, the general conditions which cause an increase or a decrease in the percentages of transformation are similar for all three. Could these opposed effects be due to random fluctuations? Since both the stimulation PURO, and CM are somewhat
and inhibition of transformation by AMD, variable in nature, the question arises whether Experimental
Cell Research 45
300
Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
these observed effects might be due to random fluctuations. To answer this problem, a number of duplicate experimental control flasks in antiserum alone were prepared on different days. Of the 138 pairs of readings of transforming cells (in 32 experiments), only twice did the difference between duplicate AS flasks reach as high as 16 per cent; in 130 out of 138 duplicate readings, the difference in extent of transformation was 10 per cent or less, V. Inhibition of the D-to-B transformation in 1 :I200 antiserum (AS) by (A) pyromycin (PIJRO) and by (B) chloramphenicol (CM); acceleration of transformation by (C) PURO and by (D) CM.
T.~RLE
Compare
conditions under which inhibition and acceleration and antibiotic, times of adding the antibiotic,
Addition of PURO or CM (hr) relative to AS
Concentrations of antibiotic used with 1:1200 AS
are obtained; concentrations and temperatures. Percentages of transformation to 51B after antiserum 8 hr
(A) 19°C 0 +3
Control ( -AS, - PURO) AS PURO (100 pug/ml) PURO (100 pg/ml) I-AS AS + PURO (100 ,ug/ml)
0 79 0 55 24 5 hr
0
Control ( -AS, -CM) AS CM (1.5 mgjml) CM (1.5 mg/ml) +AS AS t CM (1.5 mg/ml) CM (2 mg/ml) CM (2 mg/ml) +AS
0 76 0 43 3 0 0
0
Control ( -AS, - PURO) AS PURO (2.5 pg/ml) PURO (2.5 ,ug/ml) + AS
0 47 0 98
0
Control AS CM (40 CM (40 CM (40
0 15 0 64 42
(‘3 27T 0 +1!z
6: hr
((2 27°C
6 hr ("1
27°C -3
Experimental
Cell Research 45
( -AS,
- CM)
,ug/ml) pg/ml) + AS ,ug/ml) + AS
of AS
Effects of antibiotics
on transformation
in P. aurelia. I
301
in 106, 5 per cent or less. A summary of these duplicate control differences have been shown in Table II, to compare them with the extent of inhibition or stimulation produced in the experimental combinations. Thus it appears that the observed inhibition and stimulation of transformation cannot be attributed merely to experimental variations. When the experimental results, in controls or in experimental groups, are compared from day to day, some differences between experiments in the extent of transformation are of course evident; nevertheless, in general the same pattern of response is always observed. DISCUSSION
The principal modes of action of each of the three antibiotics used have been well established. (1) AMD is known to inhibit the DNA-dependent synthesis of RNA by effectively binding to regions of DNA containing guanine [IS, 351. Acs et al. [l], Revel et aZ. [36], and Honig and Rabinovitz [19] have found other modes of action which appeared in addition to the inhibition of DNA synthesis. (2) CM is an inhibitor of protein synthesis [17] and is known to act at some stage between the attachment of amino acids to sRNA and the release of the peptide chain from the ribosome (see references in Weisberger and Wolfe [46]). (3) PURO, another inhibitor of protein synthesis, acts by mimicking an aminoacyl sRNA, causing unfinished proteins to be sloughed off the ribosome [47] or releasing single ribosomes from the polyribosomal structures [25]. The inhibition or stimulation by the antibiotics of antiserum-induced transformation appears dependent upon the temporal relations between two distinct events, namely antibiotic action and the initiation of transformation. If the action of the antibiotics overlaps the start of transformation, as may be assumed to occur when high concentrations of antibiotic and of antiserum are used simultaneously, an inhibition or delay of transformation always results. If, on the other hand, induction of transformation is delayed relative to the time of addition of antibiotic, as may be assumed to be the case when antibiotics are added some hours before the addition of transforming antiserum or when slowly acting transforming conditions (dilute antiserum, low temperature) are employed, then the principal action of the antibiotics is to reinforce the transforming process. Such reinforcement is manifested by transformation sometimes starting earlier in the experimental culture than in the antiserum control, always proceeding faster in this culture (possibly because in more individuals a tentative or incomplete transformation is carried to completion [5, 8, ll]), and often reaching higher final precentage Experimental
Cell Research 45
302
Mary I,. Austin, J. Pasternak
and Bertina
M. Rudman
levels. (All three of these manifestations are evident in the results of the two experiments shown in Figs. 1 A and 1 R.) In a general way, as Sonneborn 1411 has pointed out, the antigen system of P. aurelia appears analogous to inducible and/or repressible enzyme systems of bacteria. It therefore seems reasonable to expect to find some sort of regulatory mechanism controlling the functioning of structural genes for the antigens in Paramecium similar to that controlling the syntheses of enzyme systems in bacteria [2Oj. With the present dearth of knowledge concerning the kinetics of macromolecular syntheses during induced transformation, speculations concerning the mechanism of transformation must be confined to very general hypotheses. Two theoretical suggestions will be considered here, with certain other questions and suggestions discussed further in the second paper of this series. (1) Since, as brought out above, investigators are beginning to find that, at least in in vitro preparations, AMD may have other effects than the characteristic one of inhibition of DNA-dependent RNA synthesis, the question arises as to whether the data permit concluding that the antibiotics have different basic actions in low and in high concentrations, leading to acceleration and to inhibition of transforlllation, respectively. Against this conclusion is the fact that in the two experiments shown in Table IV-A the high concentration of AMD (12.5 pg/ml), instead of inhibiting transformation, stimulated it as a result of being applied along with low temperature (19°C) and a very high dilution of antiserum (1 : 2400). Under the conditions of these experiments, transfornlation begins to appear-both in controls and in experimentals-only after about 15-21 hr, and it could be argued that by that time the init.ial high concentration of antibiotic had been reduced to a low effective concentration. This argument, however, fails to account for two other facts. First, the acceleration of transformation (in 1:2400 antiserum) was evident throughout the 48 hr during which the experiment was followed. Second, the same concentration of AMD totally inhibited transformation fol 48 hr when applied along with higer concentrations of antiserum (1: 800 and 1: 1600) at the same temperature (19’C). These facts seem virtually to eliminate the possibility that different direct actions of the antibiotics occur in dependence on their concentrations. Since this is so, to avoid what may be merely unnecessary complications, it will be assumed that each antibiotic functions in its typical manner when either inhibition or stimulation of transformation is observed. Justification for this assumption in the case of inhibition lies in the results of preliminary labeling experiments which indicate that AMD (12.5 ,ug/ml) will inhibit at Experimental
Cell Research 45
Effects of antibiotics
on transformation
in P. aurelia. I
303
least 60 per cent of the cellular RNA synthesis in P. aurelia under conditions paralleling those described in the present experiments, and that CM (200 ,ug/ml) inhibits protein synthesis by 66 per cent. Thus there is little doubt that both RNA and protein are synthesized as an essential part of antigen transformation. Further labeling experiments need to be done to help solve the problem of additional actions of the antibiotics. (2) The second theoretical suggestion may have to be revised or even discarded as more is learned about the molecular events of transformation, but at present the known facts appear to agree with it and its application makes it possible to predict in a general way whether inhibition or stimulation will result from the particular set of conditions employed. For this explanation three assumptions are necessary: (a) that the maintenance of an antigen at the surface depends upon a continuing synthesis of the existing protein; (b) that the antibiotics, AMD, PURO, and CM, block at their respective sites whatever synthesis is going on in the cell at the time of their entrance; and (c) that blockage of the synthesis of an antigen at any point, whether at the DNA-RNA level or later, will generally result in a switch to a new synthesis. Thus, only if the presence of an antibiotic coincided with the onset of the new serum-induced synthesis could it be detected as manifesting an inhibitory effect. On the other hand, if the action of the antibiotic preceded that of the new serum-induced synthesis, then it would block synthesis of the original antigen before synthesis of the new antigen began. This would reinforce the action of antiserum in blocking that synthesis and releasing new antigen synthesis, and therefore would accelerate transformation. This hypothesis thus assumes that in both inhibition and acceleration the antibiotics are working in essentially the same way, i.e., by inhibiting a synthesis. Others workers have also observed a stimulatory effect with actinomycin D [26-28, 371 and puromycin [26] on hormone-mediated and substrate-induced enzyme synthesis, and have accounted for these phenomena in various ways. Whether there is a common basis for all the observations remains to be determined. Obviously more data are needed about the kinetics of macromolecular syntheses during the various experimental conditions which have been employed. The results in this paper are in general agreement with observations to be reported in the second paper of this series on the effects of the three antibiotics on another antiserum-induced transformation and on transformations induced by patulin [3] and by acetamide (Austin, unpublished).
Experimental
Cell Research 45
304
Mary IT. Austin, J. Pasternak and Bertina
Al. Rudman
SUMMARY
Antiserum-induced transformation in Paramecium aurelia can be inhibited or stimulated by the addition of actinomycin D, puromycin, or chloramphenicol. In general, simultaneous treatment with high concentrations of both the antibiotics and the inducer (antiserum) inhibits transformation. As the concentration of antiserum is reduced while that of the antibiotic remains high or as the concentrations of both antiserum and antiobotics are reduced, points are reached where further reduction stimulates the transformation. The same low concentration of an antibiotic may inhibit the transformation if added after the antiserum yet stimulate it if added before the antiserum. Low concentrations of the antibiotic can inhibit the transformation at one temperature (31 “C) and stimulate it at another (19°C). All these seemingly paradoxical findings can be reconciled on the assumption that the antibiotics inhibit the synthesis of the existing antigen when they stimulate transformation, inhibit the synthesis of the induced antigen when they interfere with transformation. The authors are grateful manuscript.
to Dr T. M. Sonneborn for a critical reading of the REFERENCES
1. Acs, G., REICH, E. and VALANJU, S., Biochim. Biophys. Acta 76, 68 (1963). 2. ALLEN, S. L., in M. FLORKIN and R. SCHEER (eds.), Chemical Zoology, Vol. 1. New York, Academic Press, 1966. 3. AUSTIN, M. L., WID~AYER, D. and WALKER, L. M., Physiol. Zool. 29, 261 (1956). 4. BALBINDER, E. and PREER, J. R., JR., J. Gen. Microbial. 21, 156 (1959). 5. BEALE, G. H., Proc. Natl kad. Sci. 34, 418 (1948). 6. __ The genetics of Paramecium aurelia. Cambridge, Cambridge University Press, 1954. 7. --Infern. Reu. Cytol. 6, 1 (1957). 8. __ Proc. Roy. Sot. B 148, 308 (1958). 9. ~ ibid. 164, 209 (1966). 10. BEALE, G. H. and KACSER, H., J. Gen. Microbial. 17, 68 (1957). 11. BEALE, G. H. and MOTT, M. R., ibid. 28, 617 (1962). 12. BEALE, G. H. and WILKINSON, J. F., Ann. Reu. Microbial. 15, 263 (1961). 13. BISHOP, J. 0. and BEALE, G. H., Nature 186, 734 (1960). 14. DRYL, S., Exptl Cell Res. 37, 569 (1965). 15. FINGER, I., Biol. Bull. 111, 358 (1956). 16. FINGER, I., HELLER, C. and GREEN, A., Genetics 47, 241 (1962). 17. GALE, E. F. and FOLKES, J. P., Biochem. J. 53, 493 (1953). 18. GOLDBERG, I. H. and REICH, E., Fed. Proc. 23, 958 (1964). 19. HONIG, G. and RABINOVITZ, M., Science 149, 1504 (1965). 20. JACOB, F. and MONOD, J., J. Mol. Biol. 3, 318 (1961). 21. JONES, I. G., Biochem. J. 96, 17 (1965). 22. JONES, I. G. and BEALE, G. H., Nature 197, 205 (1963). 23. KIMBALL, R. F., in S. H. HUTNER (ed.), Biochemistry and Physiology of Protozoa, Vol. 3, pp. 243-275. Academic Press, New York, 1964.
Experimental
Cell Research 45
Effects of antibiotics
on transformation
in P. aurelia. I
305
24. MARGOLIN, P., Genetics 41, 685 (1956). 25. MARKS, P. A., BURKA, E. R., RIFKIND, R. and DANON, D., Cold Spring Harbor Symp. Quark Biol. 28, 223 (1963). F., Science 144, 414 (1964). 26. Moot, 27. NITOWSKY, H., GELLER, S. and CASPER, R., Fed. Proc. 23, 556 (1964). 28. POLLOCK, M. R., Biochim. Biophys. Acta 76, 80 (1963). 29. PREER, J. R., JR., Genetics 33, 349 (1948). 30. -Ann. Reu. Microbial. 11, 419 (1957). 31. ~in D. RUDNICK (ed.), Developmental Cytology, pp. 3-20. The Ronald Press, New York, 1959.
32. -33. --
J. Immunol. 83, 385 (1959). Genetics 44, 803 (1959).
34. 35.
PREER, REICH, 36. REVEL, 37. ROSEN,
J. R., JR. and PREER, L. B., J. Protozool. 6, 88 (1959). E., FRANKLIN, R. M., SHATKIN, A. J. and TATUM, E. L., Science 134, 556 (1961). M., HIATT, H. H. and REVEL, J., ibid. 146, 1311 (1964). F., RAINA, P. N., MILHOLLAND, R. J. and NICHOL, C. A., ibid. 146, 661 (1964). 38. SONNEBORN, T. M., Proc. Nat1 Acad. Sci. 34, 413 (1948).
39. -~ 40. -41.
42. 43. 44. 45. 46. 47.
Heredity 4, 11 (1950). .I. Exptl Zool. 113, 87 (1950). ~ Proc. Nat! Acad. Sci. 46, 149 (1960). STEERS, E. J., JR., Science 133, 2010 (1961). ~ Proc. Natl Acad. Sci. 48, 867 (1962). ~ Biochem. 4, 1896 (1965). VAN WAGTENDONK, W. J., VAN TIJN, B., LITMAN, R., REISNER, A. and YOUNG, Gen. MicrobioI. 15, 617 (1956). WEISBERGER, A. S. and WOLFE, S., Fed. Proc. 23, 976 (1964). YARMOLINSKY, M. B. and DE LA HABA, G. L., Proc. Nat! Acad. Sci. 45, 1721 (1959).
Experimental
M.,
J.
Cell Research 45
Inc.
Cell Research 45, 289-305 (1967)
STUDIES
ON THE MECHANISM
FORMATION I.
THE
EFFECTS
OF
ACTINOMYCIN
INDUCED
L.
AUSTIN,
OF SEROTYPIC
IN PARAMEC1UM
CHLORAMPHENICOL
MARY
289
ON
D, AN
TRANS-
AURELIA PUROMYCIN,
AND
ANTISERUM-
TRANSFORMATION’
J. PASTERNAKs
and BERTINA
Department of Zoology, Indiana University, Bloomington, Received
M. RUDMAN Ind., U.S.A.
June 13, 1966s
THE basic features of the surface antigen system of Paramecium aurelia have been described and discussed in numerous papers, e.g., in those of Sonneborn [38, 39, 411, of Beale [B-9], of Preer [30, 311, of Beale and Wilkinson [la], of Kimball [23], and of Allen [a]. Briefly, each genitally homozygous clone of P. aurelia has a repertory of serotypes (antigenic types, immobilization antigens, or surface antigens); in stock 51, syngen 4, for example, there are at least 12 non-allelic loci, each of which determines a specific antigenic type. A distinguishing feature of the serotype system in P. aurelia is that at one time in any cell only one antigenic type determined by a single gene locus is expressed despite each cell’s potential ability to determine an array of different antigenic types. Only a few exceptions to this rule of mutual exclusion have been reported [16, 241. Which particular antigenic type is expressed is not merely gene-dependent but is also influenced by the state of the cytoplasm (prior and prcscnt), by the stage in the life cycle (whether autogamy-immature or autogamy-mature) [ 141, and by various extrinsic environmental factors, such as temperature, type of culture medium, pH, salt content of the medium, homologous antiserum, radiation, enzymes, and the antibiotic patulin [3] (see reviews listed above). The ciliary antigens have been found to coat the cell surface as well as the cilia
[lo,
11, 15, 34, 451. They are known to be fibrous
heteropolymeric
r Contribution no. 793 from the Department of Zoology, Indiana University. This investigation was supported mainly by Public Health Service Research Grant no. 5-KOl-Ca 05398-03-05 from the National Cancer Institute (to M. L. A.), and in part by Genetics Training Grant PHS-GM-82-05-06 (to J. P.); and was aided by contract COO-235-21a from the Atomic Energy Commission and grant no. E-81H from the American Cancer Society to T. M. Sonneborn. a Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada. 3 Hevised version received October 10. 1966. Experimental
Cell Research 45
Mary I,. Austin, J. Pasternak
and Bertina
M. Rudman
polypeptide structures [al, 32, 441, and within a stock the different antigenic types determined by non-allelic loci have been found to possess divergent immunological and physico-chemical properties [ 13, 33, 421. Moreover, the amino acid content of the different antigens varies significantly [al, 22, 43, 441, an observation which suggests that a change from one antigenic type to another within a clone probably does not entail refolding of the existing protein structure, but more likely involves de novo protein synthesis of all or part of the “new” antigen [4, 21, 431. Sonneborn [38] was the first to demonstrate that exposure to homologous antiserum (AS) stimulated transformation, that the direction of transformation (i.e., the serotype to which the animals were transformed) could be controlled by prescribed temperatures and/or the quantity of food supplied daily, that transformations were reversible, and that certain diverse serotypes could be maintained during reproduction more or less indefinitely under identical conditions of culture. The present investigation was undertaken to enlarge our understanding of the nature of the biosynthetic processes during transformation, and to gain some insight into the mechanisms that maintain a particular antigenic type. To test the hypothesis that these processes and mechanisms inc,lude gene-controlled synthesis of the antigenic proteins, the effects of three antibiotics, actinomycin D (,4MD), puromycin (PURO), and chloramphenicol (CM), were investigated. The actions of these antibiotics are reasonably well known [1, 17-19, 35, 36, 46, 471. All three antibiotics were found to affect the transformation of serotypes; the particular one studied here was the antiserum-induced transformation of 5lD to 51R. (The different serotypes in P. aurelia are designated by capital letters, the different stocks by numbers. Since only stock 51 was used in this study, the serotypes will hereafter usually be referred to by the letter alone.) As will appear, the effects of the antibiotics vary in dependence upon their concentration, the prevailing temperature, the concentration of the homologous antiserum used to induce transformation, and the time relations between exposure to homologous antiserum and exposure to antibiotic. The interrelations among these variables provide insight into the nature of the process of serotype transformation. The most remarkable and informative aspect of the results is that each antibiotic either inhibits or stimulates serotype transformation in definite and regular dependence upon the particular combination of variables.
Experimental
Cell Research 45
Effects of antibiotics
MATERIALS
on transformation
AND
in P. aurelia. I
291
METHODS
Growth of cultures prior to experiments; selection of paramecia for testing.-Cultures were prepared for the experiments as follows. Single washed cells of P. aurelia, serotype D of stock 51, syngen 4, with genotype KK, lacking kappa, were grown up into tube cultures and maintained in 0.15 per cent cerophyl, containing 0.6-0.9 g 12H,O)/l. This medium was used 24 hr of dibasic sodium phosphate (Na,HPO,. after inoculation with Aerobacter aerogenes at which time the pH was 7.0-7.2. Enough inoculated cerophyl medium was added daily to the cultures to maintain reproduction at the rate of one fission per day [29,40]. When used for an experiment, such cultures might have passed through as many as three autogamous periods since the initial isolation, but, if selected for an experiment, a culture had at that time no more than 5 per cent of the animals in autogamy. At the start of an experiment, always 24 hr after the last feeding, the culture was usually>97 per cent pure for serotype D. Chemicals.-Puromycin dihydrochloride was purchased from Nutritional Biochemicals Corporation; chloramphenicol from Parke, Davis and Company. We acknowledge with thanks the gift of actinomycin D from Merck, Sharp and Dohme, West Point, Pa. Procedure.-Experiments were carried out as follows. From the preparatory tube cultures four aliquots of about 3000 cells in 10 or 12 ml of culture medium were put into 50 ml flasks. The culture medium and pH used for the experiments were those which in general favour the appearance and long maintenance of 51B, i.e., baked lettuce medium adjusted to a pH of about 6.8 by the use of sodium phosphate buffer. To one flask was added an antibiotic, to the second homologous antiserum, to the third both, and to the fourth neither. All flasks were brought to the same final volume, for example, by adding distilled water instead of antibiotic, or non-homologous antiserum instead of homologous antiserum. Since cultures of D at times had up to 3 per cent contamination with serotypes A, C, and/or E, antisera against these types were added along with the anti-D serum in the same non-lethal concentration as was the anti-D serum (given below). At intervals after the flasks were prepared, samples were withdrawn and tested for serotype. Animals were scored as being of a certain serotype only when they were completely immobilized by that particular antiserum [3]. Experiments were run in varying concentrations of the AS (1: 2400 up to 1: 800, none of them lethal) as well as of the antibiotics. Most of the different combinations of concentrations of AS and of antibiotic (high AS with high antibiotic, low with low, and reciprocal combinations) have been tested at 27°C with certain ones also tested at 14”, 19”, and 31°C. In general, each experiment of a kind has been run at least twice, and some repeatedly. Details will be given as the experiments are reported under Results. Inhibition and stimulation were detected operationally by comparing the percentages of transformed cells in controls lacking antibiotics with the percentage in experimentals exposed to antibiotics. Such comparisons, made at intervals during the course of an experiment, made it possible to detect effects on speed of transformation of a culture up to the maximum percentage transformed. Such effects do Experimental Cell Research 45
292
Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
not necessarily imply an effect on the speed of transformation of individual cells because the moment of initiation of the transformation processes could not be directly ascertained, and delays or accelerations in the final completed transformation of the cells (the observed phenomenon) could have been due to variations in the onset of the initiation of their transformation. Thus inhibition and stimulation of transformation of a culture took three observable forms: (1) later or earlier onset, and/or (2) retardation (deceleration) or hastening (acceleration) of transformation, and/or (3) reduction or increase in the final percentages of transformation as compared with those in the AS controls. Hence it is important to define the end point of the experiments. In general, experiments were terminated when deaths began to appear in one or more cultures or when the percentage of transformation plateaued in the controls exposed to antiserum alone, i.e., by 48 hr. Usually deaths did not occur within 48 hr and the antiserum controls exhibited maximal transformation by that time or sooner.
RESULTS Effects on the cells, other than on antigen alone and of antiserum (AS) alone
transformation,
of the antibiotics
Higher concentrations of the three antibiotics had deleterious effects on the paramecia, but lower concentrations did not. Little or no effect on fission rate was produced by concentrations of up to 1 ,ug/ml of AMD, up to 10 ,ug/ml of PURO, and up to 40 pg/ml of CM. However, division was suppressed without occurrence of deaths during three days of exposure to 2-3 pg/ml of AMD, to 25-80 pg/ml of PURO, and to 50 ,ug/ml-2.0 mg/ml of CM. Certain abnormalities in the paramecia were noted in concentrations equal to or greater than 5 rug/ml of AMD (the macronuclei tended to be enlarged or broken into 2-4 pieces), 50 ,ug/ml of PURO (the shape of some of the animals tended to be abnormal), and 500 ,ug/ml of CM (the animals tended to be large and dark). Deaths occurred on the third day in concentrations of 5-15 ,ug/ml of AMD and as early as the second day in 25 pg/ml. In PURO animals died on the third day in 100 ,ug/ml; no tests were carried in concentrations stronger than this. The strongest concentration of CM used was 2.0 mg/ml in which, as noted, no dying occurred. Antisera were used in concentrations of 1 : 800, 1 : 900, 1: 1200, and 1 : 2000 repeatedly. In addition, the concentrations of 1: 1600 (in three experiments), of 1: 1800 (in one experiment), and of 1: 2400 (in two experiments) have been tested. Except for a progressive reduction in mobility as the concentration increased, no deleterious effects on the paramecia were produced by any of these concentrations of antiserum. Experimental
Cell Research 45
Effects of antibiotics Eflects on antigen one temperature
transformation
on transformation of varying
in P. aurelia. I
293
the serum concentration
alone at
At each of the four temperatures (14”, 19”, 27”, and 31°C) increasing the serum concentration alone results in general in a progressive increase in the rapidity with which the percentages of transformed cells increase in a culture. This fact is made clear by the following mean percentages of transformation in different concentrations of antiserum at 19°C. After 9 hr of exposure in antisera 1: 2400, 1: 2000, 1: 1600, 1: 1200, and 1: 800, the mean percentages of transformation in this order were 0, 5, 5, 77.5, and 89.5. The maximum percentages of transformation obtained in these concentrations when the experiments ended at 48 hr were, in the same order, 45, 72 (at 24 hr), 78, 100, 98. Similarly, increasing percentages of transformed paramecia were obtained for antiserum concentrations of 1 : 2000, 1 : 1200, and 1 : 800 at both 27” and 31°C. Although the differences described can be slight in adjacent concentrations, Table I shows that they are quite marked in the more widely separated concentrations of 1: 2000 and 1: 800. Effects on antigen transformation antiserum concentration
of varying
the temperature
alone at one
The speed of transformation is also increased by increasing the temperature while keeping the antiserum concentration the same. The temperature effect is clearly shown by comparing the 6-hr results for the 1: 800 concentration (0, 40.5, and 100 per cent transformed) and the 8-hr results for the 1: 2000 concentration (2, 22, and 85 per cent transformed) at the three temperatures used (Table I). The relative effects of temperature transformation
and of antiserum
concentration
on speed of
When the relative effects of temperature and of antiserum concentration on speed of transformation are compared (Table I), it is clear that the temperature effect predominates, since at 31” the paramecia in almost the lowest concentration used (1: 2000) transform at a faster rate than they do in the highest concentration at either of the other two temperatures. This strong influence of temperature on the speed of transformation cannot be attributed to a general tendency of B to emerge at high temperatures, because in stock 51 serotype B is predominantly a low-temperature type [38] and because D control cells without antiserum, under the conditions and duration of these 19 - 671812
Experimental
Cell Research 45
Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
experiments, never transformed to B on exposure to increased temperature alone. Thus the temperature must act, not in directing the transformation to B, but in speeding up this transformation. Presumably this speeding up is accomplished through the general effect of temperature on rates of reactions. Acquiring control of the speed of transformation in the ways described by adjusting antiserum concentration and temperature has proved to be of importance in the experiments with the three antibiotics, AMD, PURO, and CM. Effects of AMD on antigen
transformation
Table II shows in summary form the occurrence and general extent of inhibition or stimulation of the D-to-B transformation produced by combinations of AS with 0.5 ,ug/ml of AMD. As we have seen, an antiserum concentraTABLE
I. Speed of transformation of serotype D to B at different and in different concentrations of antiserum.
temperatures
This is a composite table with each value representing the mean of transformation obtained in all experiments for a particular antiserum concentration, temperature of observation. The number of experiments used in calculating each mean is shown in following the percentage figure; the maximum number shown in a parenthesis in represents the total number of experiments run with that combination of antiserum tion and temperature. As is evident, observations were not made in all experiments Time of scoring tests W) 3 4 5 6 7 8 9 10 12 14 16
Concentrations At 19°C --1:soo
Experimental
1:800
At 31°C 1:2000
r--7 1:soo
1:2000
60 (2) 0 (2) 0 (3) 64 (2) 89.5 (1) 97.5 (1) 93 (3) 100 (2)
0 (2) 0.5 (2)
2 (2) 5 (2) 13 (3) 28 (3)
0 (5) 14 (9) 40.5 (9) 76.5 (9)
0 (2) 0 (2) 8 (13) 18.5 (11)
87 (3) 94 (3) 100 (3) 99.5 (3)
11 (2) 54 (2) 81 (2) 82 (2)
93 (6) 97 (8) 99 (‘3) 99 (6)
22 (10) 50 (11) 52.5 (13) 66 (13)
100 (3)
85 (2)
66 (2)
-
78.5 (2)
65 (3) 89.5 (5) 96.5 (3)
18 20 24 48
of 51D-antiserum At 27°C
1:2000
percentages and time parentheses any column concentraat all hours.
98 (3) 98 (3)
72 (3)
Cell Research 45
100 (7)
79.5 (9) 88 (3)
97.5 (3)
96
(2)
93
(2)
Effects of antibiotics on transformalion
295
in P. aurelia. Z
tion of I : 800 and a temperature of 31°C both hasten the onset of transformation. When AbfD (0.5 ,q/ml) is used simultaneously with these factors that cause rapid transformation, inhibition always results. When a combination of conditions are employed which now slow down ~ransformt~on in the AS controls, a progressively greater stimulatory effect results on the combinations of AMD and serum. When this concentration of AMD is used in combination with 1: 1200 AS at 27”C, tral~sformation appears to be usualIS inhibited, hut sometimes stimulated; the conditions here are apparently ~~~~~~~~ o( e#j%Trs oic‘ ~~~~t~~~~~~ D (0.5 ~~~~~~ irr ColttbiRatiorl with temperctture in inhibiting UPstimulating transformation of D to B induced by difrerent concentrations of antiserum (AS).
TABLE If.
The numbers of readings falling within 3 classes of percentages of inbibitition or stimulation (O-10%, 11X1-50%, and 50,1-100X) during transformation are shown. The fraction represents those experimentsof the total number In which the particular effect (inhibition or stimulation) was found under each set of conditions.
O-10 10-50 50-100
31°C x-w O--l0 10-50 50-100 O-10 IO-50 50-100
96Classes.. ,
a-10 IO-50 50-100 7
f:SOO
13
0 3 3 ‘Inhibition In 2/2
1 5 8 Inhibition In 3/3
27°C.
19°C
14OC 1
AS cont.
0
?Ximulat.ion in 7/7 expts
0 5 7 Inhibition in 212
1:soo
a 8 14 Stimulation in 6/6 expls
No test
2 7 0 Inhibition in 2/Z
1:12oa
a 4 a Stimuiation in 212 expts
3 3 0 StimuIatian in 212
0 2 (stim.) 0 a 5 Inhibitjon in 2.513
0 8 4 Inhibition in 212
1:2aoo
No test
6 0 1 Stimufation in Z/2
*5 10 0 Stimulation in 4j4
4 5 0 Inhibition in 2p
Differences between duplicate AS-controIs’
7
0
0
7 readings in 4 expts
16
1
0
17 readings in 4 expts
62
5
0
67 readings in 14 expts
hro test
45
2
0
47 readings in 10 expts
a See paragraph on Procedure for three ways in which stimulation or inhibition may be manifested. * C&e experiment in 1: 1800 antiserum inehtded here with three in 3 :2000 antiserum. ’ C)uplicate antiserum tests of the D-to-B transformation have been carried in a total of 32 experiments run at different times, with a totar of 138 readings made during transformation. Different ~on~e~tra~~onsof AS have not been separated in this summary, since there was no correlation between the concentration and the degree of difference in transformation percentages of the paired AS-controls.
296
Mary L. Austin, J. Pasternak
and Bertha
M. Rudman
borderline. However, when it has been used in experiments with 1 : 1200 AS at 19” and 14°C and with 1: 2000 at both 19” and 27”C, transformation has always been stimulated. In other words, the antibiotic stimulates or inhibits the transformation brought about by antisermn, depending upon the conditions employed in the experiment. When the consistency of these relations of temperature and AS concentration became apparent, it was predicted that stimulation with 1: 800 AS might be obtained at a temperature lower than 19°C. When this high concentration of AS was tested at 14°C with 0.5 pg/mI of AMD, stim~~lation did, in fact, result, as Table II shows. Table III shows specific results of some experiments in which transformaTABLE III.
Experimerit
la.a 1.
Inhibitory effect of acti~lomyci~ D (A~~~) on the tra~s~orI~ation of 5lD to 51 B in 1:800 antiserum (AS) at 19°C. Addition of AMD (hr) relative to that of AS
-3 0 +3 10
Presence of AS and concentrations of AMD (~g/ml)
Percentages of transformation to 518
Control ( -AS, - AMD) AS AMD (12.5) AMD (12.5) -I-AS AMD (12.5) i AS AS s AMD (12.5) AS -i-A&ID (12.5)
20 hr 0 95 0 0 0 2 7x
48 hr 0 97 0 Dead 14 40 97
0
Control ( -AS, -ARID) AS AMD (5) AMD (5) -I-AS AMD (1) AMD (1) -t-AS
13 hr 0 98 0 0 0 2
24 hr 0 98 0 1.5 0 18
48 hr 0 98 0 9 0 60b
0
Control ( -AS, - AMD) AS AMD (0.5) AMD (0.5) + AS
14 hr 0 100 0 0
24 hr 0 100 0 0
48 hr 0 96.5 0 67’
2.
0
3.
a Expt. la run at different time from Expt. 1; controls of la behaved like those of 1. ’ Includes 16.5 % of types other than B. ’ Includes 41 X of types other than Il. Experimental
Cell Research 45
Effects of antibiotics
on transformation
297
in P. aurelia. I
tion was inhibited. The antiserum concentration was 1: 800, the temperature 19”C, and the AMD concentrations employed were 12.5, 5, 1, and 0.5 ,ug/ml. When 12.5 ,ug/ml (Table III-l) was added simultaneously with, 3 hr before, and 3 and 9 hr after the antiserum, AMD proved to be a potent inhibitor, although, as the table shows, the later it was added to the antiserum, the less inhibition of transformation resulted. Similarly a concentration of 5 ,ug/ml of AMD (Table 111-2) gave a drastic inhibition of transformation through 48 hr. The lower concentrations of 1 ,ug/ml (Table 111-2) and of 0.5 ,ug/ml (Table 111-3) also reduced the amount of transformation significantly through the first day, but in the second 24 hr animals exposed to these concentrations transformed substantially, although they failed to attain the control level by the end of 48 hr of observation. TUI.E
IV.
Combinations of antiserum (AS) and actinomycin D (AMD) that accelerate the AS-induced transformation of 5lD to 51 B. Part A shows effects of using a high concentration of AMD with a very low concentration of AS from 0 time; part B shows effects of using a low concentration of AMD 3 hr before a high concentration of AS. Compare combinations that accelerate with those that inhibit transformation.
Addition of AMD (hr) relative to AS
Percentages of transformation to 51B after antiserum
Concentrations of AMD @g/ml) and of AS
24 hr Expt
(-4) 19°C 0 0 0
Control ( -AS, -AM A4MD (12.5) AS (1: 800) AS (1: 800) + AMD AS (1: 1600) AS (1:1600)+AMD AS (1: 2400) AS (1:2400)+AMD
D)
1
Expt
0 0 95 0 64 44 14 26
2
0 0 100 0 56 0 20 88 8 hr
Expt
(B) 19°C -3 0 +3
Control ( -AS, AMD (0.5) AS (1: 800) AMD i-AS AMD+AS AS+AMD
- AMD)
1
Expt
0 0 65 93 0 Experimental
2
0 0 78 0 0 Cell Research 45
Mary L. Austin, J. Pasternak
298
and Bertina
M. Rudman
Table IV shows some examples of stimulation of transformation at 19”C, in contrast with inhibition in the same experiments, when AMD was used in two widely separated concentrations. Table IV-A shows the results of two experiments with a high concentration of AMD, 12.5,~g/ml. These demonstrate that, although this high concentration drastically inhibits the transformation produced by 1: 800 antiserum and less drastically inhibits that brought about by 1: 1600 antiserum, it can stimulate transformation when the initial transformation-inducing stimulus is very weak, as it is in 1: 2400 antiserum; in other words, when the progress of transformation in the controls is quite slow. Table IV-B shows that stimulation of transformation can also be obtained when 0.5 ,ug/ml of AMD is given 3 hr prior to exposure to 1:800 AS at 19°C. We have previously pointed out that a third method of slowing down transformation in the controls and of obtaining stimulation is possible, i.e., by lowering the temperature (Table II).
4
5
6
7
8
9
IO
II
12 HOURS
AFTER
ANTISERUM
Fig. l.-The effects of actinomycin D on antiserum-induced transformation in P. aurelia at 27°C. (A) This figure shows the effects of 0.5 ,ug/ml of actinomycin D on the 51 D-to-51B transformation induced with two concentrations of homologous antiserum in one experiment. Transformation percentages in cells treated with homologous antiserum alone are shown for concentration 1: 800 as 0-0, for 1:2000 as A-A; with actinomycin D alone, as W-W; with culture fluid alone, as 0-n; and with actinomycin D in combination with 1:800 antiserum, as O-O, with 1:2000 antiserum as A-A. (B) This figure shows the results of a duplicate experiment. The experimental conditions and the symbols used are the same as those described under A. Experimental
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Effects of anfibiofics
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in P. aurelia. I
299
Figs. 1 A and 1 B show graphically the results of two experiments in which both inhibition and stimulation of transformation were obtained by the same concentration of 0.5 pg/ml of AMD, this time at 27°C and combined with 1:SOO and with 1: 2000 antiserum. The usual controls were run in flasks containing culture fluid alone, AMD alone, and each of the antiserum concentrations alone. With the faster transformation, obtained by the use of antiserum 1:800, AMD inhibited transformation, vvhereas the same concentration of AMD stimulated transformation of the more slowly transforming group in antiserum 1: 2000. It should be noted that AMD treatment alone seldom caused serotypic changes in the first 48 hr. Out of the total of 50 experiments run to test the effect of AMD (with concentrations ranging from 0.125 pg/ml to 25 pg/ml) in only two instances, both from the same experiment, was there any transformation in AMD alone before the end of 48 hr. In these cases, D cells transformed after 30 hr 26.5 per cent to B and 70 per cent to N when treated with 3 pg/ml of ,4MD and 100 per cent to N when treated with 6 pg/ml of AMD. In general, such transformation occurred only on the third day and only in the lower concentrations (2-6 pg/ml), if it occurred at all. It should also be noted that in four experiments low concentrations of AMD (Q 5 pg/ml) used in conjunction with non-homologous antiserum never stimulated transformation above that in the AS control. Efecfs of PURO and CM on antigen
transformation
Similarly designed experiments were performed to ascertain the effects of PURO and of CM on antiserum-induced antigenic transformation. PURO (100 ,ug/ml) and CM (2 0.5 pg/ml) inhibit the transformation of D to B in 1: 1200 concentration of homologous antiserum. Lower concentrations of PURO (2.5 pg/ml) and of CM (40 pg/ml) combined with antiserum concentrations of 1: 800 and 1: 1200 stimulate the transformation, the stimulation being greater in the latter concentration than in the former. The results for the 1: 1200 concentration are shown in Table V. Thus it is clear that, even though the three antibiotics act at different sites in the cell, the general conditions which cause an increase or a decrease in the percentages of transformation are similar for all three. Could these opposed effects be due to random fluctuations? Since both the stimulation PURO, and CM are somewhat
and inhibition of transformation by AMD, variable in nature, the question arises whether Experimental
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Mary L. Austin, J. Pasternak
and Bertina
M. Rudman
these observed effects might be due to random fluctuations. To answer this problem, a number of duplicate experimental control flasks in antiserum alone were prepared on different days. Of the 138 pairs of readings of transforming cells (in 32 experiments), only twice did the difference between duplicate AS flasks reach as high as 16 per cent; in 130 out of 138 duplicate readings, the difference in extent of transformation was 10 per cent or less, V. Inhibition of the D-to-B transformation in 1 :I200 antiserum (AS) by (A) pyromycin (PIJRO) and by (B) chloramphenicol (CM); acceleration of transformation by (C) PURO and by (D) CM.
T.~RLE
Compare
conditions under which inhibition and acceleration and antibiotic, times of adding the antibiotic,
Addition of PURO or CM (hr) relative to AS
Concentrations of antibiotic used with 1:1200 AS
are obtained; concentrations and temperatures. Percentages of transformation to 51B after antiserum 8 hr
(A) 19°C 0 +3
Control ( -AS, - PURO) AS PURO (100 pug/ml) PURO (100 pg/ml) I-AS AS + PURO (100 ,ug/ml)
0 79 0 55 24 5 hr
0
Control ( -AS, -CM) AS CM (1.5 mgjml) CM (1.5 mg/ml) +AS AS t CM (1.5 mg/ml) CM (2 mg/ml) CM (2 mg/ml) +AS
0 76 0 43 3 0 0
0
Control ( -AS, - PURO) AS PURO (2.5 pg/ml) PURO (2.5 ,ug/ml) + AS
0 47 0 98
0
Control AS CM (40 CM (40 CM (40
0 15 0 64 42
(‘3 27T 0 +1!z
6: hr
((2 27°C
6 hr ("1
27°C -3
Experimental
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( -AS,
- CM)
,ug/ml) pg/ml) + AS ,ug/ml) + AS
of AS
Effects of antibiotics
on transformation
in P. aurelia. I
301
in 106, 5 per cent or less. A summary of these duplicate control differences have been shown in Table II, to compare them with the extent of inhibition or stimulation produced in the experimental combinations. Thus it appears that the observed inhibition and stimulation of transformation cannot be attributed merely to experimental variations. When the experimental results, in controls or in experimental groups, are compared from day to day, some differences between experiments in the extent of transformation are of course evident; nevertheless, in general the same pattern of response is always observed. DISCUSSION
The principal modes of action of each of the three antibiotics used have been well established. (1) AMD is known to inhibit the DNA-dependent synthesis of RNA by effectively binding to regions of DNA containing guanine [IS, 351. Acs et al. [l], Revel et aZ. [36], and Honig and Rabinovitz [19] have found other modes of action which appeared in addition to the inhibition of DNA synthesis. (2) CM is an inhibitor of protein synthesis [17] and is known to act at some stage between the attachment of amino acids to sRNA and the release of the peptide chain from the ribosome (see references in Weisberger and Wolfe [46]). (3) PURO, another inhibitor of protein synthesis, acts by mimicking an aminoacyl sRNA, causing unfinished proteins to be sloughed off the ribosome [47] or releasing single ribosomes from the polyribosomal structures [25]. The inhibition or stimulation by the antibiotics of antiserum-induced transformation appears dependent upon the temporal relations between two distinct events, namely antibiotic action and the initiation of transformation. If the action of the antibiotics overlaps the start of transformation, as may be assumed to occur when high concentrations of antibiotic and of antiserum are used simultaneously, an inhibition or delay of transformation always results. If, on the other hand, induction of transformation is delayed relative to the time of addition of antibiotic, as may be assumed to be the case when antibiotics are added some hours before the addition of transforming antiserum or when slowly acting transforming conditions (dilute antiserum, low temperature) are employed, then the principal action of the antibiotics is to reinforce the transforming process. Such reinforcement is manifested by transformation sometimes starting earlier in the experimental culture than in the antiserum control, always proceeding faster in this culture (possibly because in more individuals a tentative or incomplete transformation is carried to completion [5, 8, ll]), and often reaching higher final precentage Experimental
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Mary I,. Austin, J. Pasternak
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M. Rudman
levels. (All three of these manifestations are evident in the results of the two experiments shown in Figs. 1 A and 1 R.) In a general way, as Sonneborn 1411 has pointed out, the antigen system of P. aurelia appears analogous to inducible and/or repressible enzyme systems of bacteria. It therefore seems reasonable to expect to find some sort of regulatory mechanism controlling the functioning of structural genes for the antigens in Paramecium similar to that controlling the syntheses of enzyme systems in bacteria [2Oj. With the present dearth of knowledge concerning the kinetics of macromolecular syntheses during induced transformation, speculations concerning the mechanism of transformation must be confined to very general hypotheses. Two theoretical suggestions will be considered here, with certain other questions and suggestions discussed further in the second paper of this series. (1) Since, as brought out above, investigators are beginning to find that, at least in in vitro preparations, AMD may have other effects than the characteristic one of inhibition of DNA-dependent RNA synthesis, the question arises as to whether the data permit concluding that the antibiotics have different basic actions in low and in high concentrations, leading to acceleration and to inhibition of transforlllation, respectively. Against this conclusion is the fact that in the two experiments shown in Table IV-A the high concentration of AMD (12.5 pg/ml), instead of inhibiting transformation, stimulated it as a result of being applied along with low temperature (19°C) and a very high dilution of antiserum (1 : 2400). Under the conditions of these experiments, transfornlation begins to appear-both in controls and in experimentals-only after about 15-21 hr, and it could be argued that by that time the init.ial high concentration of antibiotic had been reduced to a low effective concentration. This argument, however, fails to account for two other facts. First, the acceleration of transformation (in 1:2400 antiserum) was evident throughout the 48 hr during which the experiment was followed. Second, the same concentration of AMD totally inhibited transformation fol 48 hr when applied along with higer concentrations of antiserum (1: 800 and 1: 1600) at the same temperature (19’C). These facts seem virtually to eliminate the possibility that different direct actions of the antibiotics occur in dependence on their concentrations. Since this is so, to avoid what may be merely unnecessary complications, it will be assumed that each antibiotic functions in its typical manner when either inhibition or stimulation of transformation is observed. Justification for this assumption in the case of inhibition lies in the results of preliminary labeling experiments which indicate that AMD (12.5 ,ug/ml) will inhibit at Experimental
Cell Research 45
Effects of antibiotics
on transformation
in P. aurelia. I
303
least 60 per cent of the cellular RNA synthesis in P. aurelia under conditions paralleling those described in the present experiments, and that CM (200 ,ug/ml) inhibits protein synthesis by 66 per cent. Thus there is little doubt that both RNA and protein are synthesized as an essential part of antigen transformation. Further labeling experiments need to be done to help solve the problem of additional actions of the antibiotics. (2) The second theoretical suggestion may have to be revised or even discarded as more is learned about the molecular events of transformation, but at present the known facts appear to agree with it and its application makes it possible to predict in a general way whether inhibition or stimulation will result from the particular set of conditions employed. For this explanation three assumptions are necessary: (a) that the maintenance of an antigen at the surface depends upon a continuing synthesis of the existing protein; (b) that the antibiotics, AMD, PURO, and CM, block at their respective sites whatever synthesis is going on in the cell at the time of their entrance; and (c) that blockage of the synthesis of an antigen at any point, whether at the DNA-RNA level or later, will generally result in a switch to a new synthesis. Thus, only if the presence of an antibiotic coincided with the onset of the new serum-induced synthesis could it be detected as manifesting an inhibitory effect. On the other hand, if the action of the antibiotic preceded that of the new serum-induced synthesis, then it would block synthesis of the original antigen before synthesis of the new antigen began. This would reinforce the action of antiserum in blocking that synthesis and releasing new antigen synthesis, and therefore would accelerate transformation. This hypothesis thus assumes that in both inhibition and acceleration the antibiotics are working in essentially the same way, i.e., by inhibiting a synthesis. Others workers have also observed a stimulatory effect with actinomycin D [26-28, 371 and puromycin [26] on hormone-mediated and substrate-induced enzyme synthesis, and have accounted for these phenomena in various ways. Whether there is a common basis for all the observations remains to be determined. Obviously more data are needed about the kinetics of macromolecular syntheses during the various experimental conditions which have been employed. The results in this paper are in general agreement with observations to be reported in the second paper of this series on the effects of the three antibiotics on another antiserum-induced transformation and on transformations induced by patulin [3] and by acetamide (Austin, unpublished).
Experimental
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Mary IT. Austin, J. Pasternak and Bertina
Al. Rudman
SUMMARY
Antiserum-induced transformation in Paramecium aurelia can be inhibited or stimulated by the addition of actinomycin D, puromycin, or chloramphenicol. In general, simultaneous treatment with high concentrations of both the antibiotics and the inducer (antiserum) inhibits transformation. As the concentration of antiserum is reduced while that of the antibiotic remains high or as the concentrations of both antiserum and antiobotics are reduced, points are reached where further reduction stimulates the transformation. The same low concentration of an antibiotic may inhibit the transformation if added after the antiserum yet stimulate it if added before the antiserum. Low concentrations of the antibiotic can inhibit the transformation at one temperature (31 “C) and stimulate it at another (19°C). All these seemingly paradoxical findings can be reconciled on the assumption that the antibiotics inhibit the synthesis of the existing antigen when they stimulate transformation, inhibit the synthesis of the induced antigen when they interfere with transformation. The authors are grateful manuscript.
to Dr T. M. Sonneborn for a critical reading of the REFERENCES
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