- Email: [email protected]
Effects of puromycin aminonucleoside on growth and chloroplast development of Euglena gracilis
Experimental
Cell
Research
EFFECTS ON GROWTH
47, 237-245
OF PUROMYCIN AMINONUCLEOSIDE AND CHLOROPLAST DEVELOPMENT EUGLENA GRACILIS M.
Department
237
(1967)
of Biology,
OF
I. SELSKY
Brooklyn
College,
Received
November
Brooklyn,
New
York
11210,
U.S.A.
21. 1966
b!h~r studies of the factors affecting chloroplast development in Euglena gracilis have been made in recent years and a well-documented revielv on this subject has recently appeared [18]. Much evidence has accumulated to suggest that the chloroplast of Euglena with its unique type of DNA4 [5, 12, 171, and its specific type of ribosome (70 S) [4, 71, may indeed function as a self-sufficient unit of biosynthesis within the cell. Smillie et al. [20] demonstrated that under certain conditions chloramphenicol could preferentially inhibit chloroplast protein synthesis of autotrophic cells while not affecting growth rate of heterotrophic Euglena. Vasquez [23] later showed that chloramphenicol preferentially binds to 70 S ribosomes and Anderson and Smillie [l] correlated this finding with the site of its effect in Euglena, the chloroplast ribosome. It was of interest to know, however, if one could affect the cytoplasmic synthesis systems of the cell without markedly affecting the syntheses localized in the chloroplasts. With this in mind we tested the effects of the aminonucleoside of puromycin (AN) on cells of EugZena. A brief account of some of these findings has been presented elsewhere [19]. The effects of AN have been shown by several investigators [6, 9, 15, 161 to be different from those of the parent compound, puromycin. The primary effect of AN is thought to be an inhibition of RNA synthesis, with most marked inhibition of ribosomal-RNA synthesis. In some systems protein synthesis is not directly inhibited. Farnham and Dubin [B] suggest that the effects of AN may be due to its acting as an analogue of uncharged S-RNA, while Dickie et al. [6] hypothesize that AN acts as a false feedback inhibitor of adenine biosynthesis.
MATERIALS
AND
METHODS
Axenic cultures of Euglena gracilis, var. bacillaris, and the Z strain were grown on a medium adjusted to pH 7.4, which had butanol and acetate as its sole carbon Experimental
Cell
Research
47
238
M.
I. Selsky
sources [ll]. Dark-adapted Euglena were obtained by growing cells in light-proof flasks and transferring them several times in a dark room equipped with a green safe-light. Light-grown cells and cells that were developing chloroplasts (greening cells) were maintained under fluorescent lighting with an intensity of about 250 foot-candles. Inocula of late log phase cells (light or dark-grown cells of the bacillaris variety, or light or dark-grown cells of the Z strain) were added to 50 ml erlenmeyer llasks, which contained O-500 /“g/ml of AN (Nutritional Biochemicals Corp.) dissolved in 10 ml of medium. These cells were grown at 26°C in the light with occasionalshaking. At various intervals aliquots were removed for cell counts, total chlorophyll determination and plating. Total cell number was determined by counting in triplicate with a hemocytometer counting chamber in early experiments, and with a Coulter Counter, Model B (Coulter Electronics Corp.) in later experiments. Viability was determined by plating cells [14] after drug treatment on medium without the antibiotic, and incubating the plates in the light. Total chlorophyll was determined by the method of Arnon [2]. EugIena cells of the Z strain used for electron microscope studies were grown in the dark on pH 7.4 medium, washed twice in 0.05 M Tris, pH 7.4, and resuspended in Tris buffer with O-500 pg/ml AN added. These cultures were placed in the light and harvested after four days. The cells were fixed in 1 per cent osmium tetroxide, pH 7.5, buffered with Verona1 acetate (cells treated with 100 and 500 ,ug/ml AN), or in 64 per cent glutaraldehyde and 1 per cent osmic acid (normal, untreated cells). All cells were embedded in Maraglas (Polyscience Co.). Sections of 300-400 A thicknesswere prepared on a Porter-Blum ultra microtome (Model MT2) with a diamond knife, and stained with uranyl acetate and lead. An RCA microscope, Model EMUSG, was used. RESULTS Fig. 1 illustrates the effect of AN on cultures of light-grown and darkadapted cells of the bacillaris variety and the Z strain when grown in the light. The data plotted are from one of three similar experiments performed, and indicate total cell number over a nine day period. It can be seen that AN does not affect growth rate at a dose of 10 rug/ml. However, marked inhibition of growth rate is evident at levels of AN of 100 pg/ml or more in all the cultures. At 50 ,ug/ml, AN effects seem to be intermediate. Growth rate inhibition caused by AN was noted for all four cell types tested, the effect being more severe with greening cells. It should be noted that in all cultures tested division does not cease abruptly upon exposure of the cells to AN, but several cell doublings can occur before the inhibitory effect is observed. At the same time that cell counts were made, samples were plated. Colonies which formed from cells that were treated at 50-100 pg/ml AN appeared later than colonies from untreated cells and were much smaller in size. Experimental
Cell Research
47
ilminonucleoside
effects in Euglena
gracilis
239
Since the plating was done on media without AN, this lag in colony formation indicated that the inhibitory effect of AN on growth was probably reversible. Some loss of viability was noted, however, when cells treated with 200-500 pg/ml AN were plated. All cells from treated cultures that did form colonies formed green colonies. Even though AN markedly inhibited growth rate for all cell types tested, dark-adapted cultures exposed to AN in the light became green. Total chlorophyll/cell levels were determined for such cells after various periods of light exposure. Fig. 2 shows data obtained from an experiment with cells of the bacillaris variety. These cells were forming chloroplasts in the light in the presence of AN dissolved in growth medium. If these data are compared with the pertinent growth data (Fig. 1) it will be seen that there is an
Fig. l.-Effect of aminonucleoside Z-LT, Z-DK refer to light-grown strain, respectively. The numbers
on growth rate of E. gracilis in the light. B-LT, BPDK and and dark-adapted cells of the bacillaris variety and the Z to the right of the curves refer to aminonucleoside level @g/ml). Experimental
Cell
Research
47
240
ill. I. Selsky
accumulation of chlorophyll in cells treated with large amounts of AN just at the time when growth rate levels off and becomes stationary (between days 3 and 5). On the ninth day of light exposure, when a slight increase in cell number occurs, the chlorophyll/cell level drops. This is not unexpected since
Fig. light
2.-Total chlorophyll levels in cells of dark-adapted E. gracilis var. in the presence of aminonucleoside. Drug levels indicated to the right
hacillaris grown of curves.
in the
greater chlorophyll accumulation occurs when growth rate decreases [21]. The untreated cells, which are actively dividing for live days, show a steady rise in chlorophyll level, although never reaching the level of the treated cultures in this experiment. In order to determine the effect of AN on chlorophyll formation and chloroplast development without concomitant cell growth we next tested dark-adapted cells placed in the light under conditions that allowed little if any cell division. Dark-adapted cells were starved in the dark on 0.05 M Tris, pH 7.4 for four days and then resuspended in the buffer plus L4N (O-500 pg/ml). The cells were then placed in the light and samples were taken at intervals. Although cell number was essentially constant under these conditions, it was noted that the amount of total chlorophyll/cell increased in all cultures with time in the light (Table I). No chlorophyll was detected in any cells at “zero” time, but higher levels were found after longer light exposures. Cells treated at 100 pg/ml AN accumulated levels of chlorophyll/ cell similar to untreated cells. Somewhat lower levels of chlorophyll/cell were observed in this experiment for cells treated at 200 pg/ml. Fig. 3, with Experimental
Cell Research
47
Aminonucleoside
effecfs in Euglena
gracilis
241
data from chlorophyll
a different experiment with non-growing cells, illustrates that accumulated even in cells treated at a very high AN level (500
P&4* In order chloroplast
to ascertain structures,
whether electron
chlorophyll microscope
levels were indicative of formed studies were made of cells that
TABLE I. Effect of aminonucleoside on chlorophyll accumulation in nongrowing, dark-adapted Euglena gracilis, var. bacillaris, placed in the 1ight.a AN (wWm1) Hr in light 0 6 24 48 72 96 120 144 168 216
0 Cells/ml 9.8 x lo6 1.1 x 10' 1.13 x 10’ 1.13 x 10’ 1.2 x 10’ 1.1 x 10’ 9.6 x lo6 8.3 x lo6 8.6 x 106 9.5 x 10”
100 Chlor.
b/Cell
0.0 0.33 3.58 8.50 10.46 13.30 14.60 20.10 20.80 21.90
a Figures represent average of three b Expressed as picagrams (pg) total
Cells/ml 9.1 x 106 9.1 x 106 9.0 x 106 9.0 x 106 9.7 x 10s 1.01 x 10’ 1.0 x 10’ 7.2 x lo6 8.2 x log 7.9 x 106
200 Chlor.
b/Cell
0.0 0.42 2.88 9.24 12.60 13.70 13.50 20.60 20.10 23.70
Cells/ml 1.4 1.2 9.6 9.6 8.2 9.3 9.7 7.2 7.2 8.0
x x x x x x x x x x
Chlor.
10’ 10’ 10” lo6 lo6 106 106 lo6 lo6 lo6
b/Ce[l
0.0 0.29 1.46 6.17 10.50 11.20 10.50 16.50 17.90 17.80
determinations. chlorophyll/cell.
greened under non-growing conditions in the presence of AN. Chloroplasts with normal lamellar structures, similar to those observed in untreated cells, mere seen in dark-adapted cells of the Z strain that were exposed to light in the presence of 100 ,ug/ml AN dissolved in Tris buffer, pH 7.4 (Figs. 4 and 5). Cells treated at a very high level of AN (500 pg/ml) possessed abnormal chloroplasts. In some instances these chloroplasts showed many unfused discs (Fig. S), while in other cells the chloroplasts appeared to have incomplete or unusual lamellar systems (Fig. 7). These chloroplasts appeared somewhat similar to those reported by Ben-Shaul et al. [3] for cells grown at 7 foot-candles of light. The 500 pg/ml AN-treated cells differ from the latter, however, for they possess fairly normal chlorophyll/cell levels. It appears that at very high AN levels some stage in the final development of the chloroplast from the proplastid may be inhibited even though chlorophyll accumulation and concomitant early protein synthesis are relatively insensitive to the drug. I6
-
671817
Experimental
Cell Research
47
242
M. I. Selsky
0
10
50
100
200
500
pgm AMINONUCLEOSIDE/ml
Fig. 3.-Total chlorophyll levels in cells of dark-adapted, non-dividing exposed to light in the presence of aminonucleoside. The bar graphs level refer to chlorophyll/cell amounts after 0, 3 and 7 days in the light.
E. gracitis var. bacillaris for each aminonucleoside
DISCUSSION
From the data reported it can be concluded that AN can markedly affect growth rate of Euglena, while not strongly interfering with accumulation of chlorophyll and the development of a chloroplast in a greening cell, That cells accumulate chlorophyll is indicative of the fact that they are synthesizing chloroplast protein [13, 21, 221. Examination of the electron micrographs presented here shows that normal chloroplast lamellar systems are formed in cells treated at levels of AN sufficient to markedly inhibit growth (100 pg/ml). If one accepts the postulate that a specific control mechanism for the synthesis of chloroplast protein exists in Euglena, which Eisenstadt and Brawerman [4, 81 report may be localized on chloroplast ribosomes, then these findings indicate such a control mechanism may be refractory to AN in the intact cell. Since the major effect of AN is believed to be concerned with inhibition of ribosomal-RNA synthesis [lo], one hypothesis presented to explain our observations is that the synthesis of chloroplast ribosomal-RNA may not be greatly affected by AN. Thus the major site of inhibition would be the ribosomal-RNA of the cytoplasmic ribosome. It must be noted, however, that it is still not known if AN affects synthesis of ribosomal-RNA for 70 S, 80 S, or both type of ribosome. Euglena chloroplast ribosomes have been shown to be of the 70 S type, while those of the cytoplasm are of the 80 S variety
Figs. 4-7.-Sections of dark-adapted E. gracitis, Z strain, exposed to light for 4 days in the presence or absence of aminonucleoside dissolved in buffer. Legend: D, disc; L, lamella; OG, osmophilic granule; P, paramylum. Marker indicates 1 p; Fig. 4, Chloroplast structure of cells exposed to light in the absence of aminonucleoside; Fig. 5, Chloroplast structure of cells exposed to light in the presence of 100 pg/ml aminonucleoside; Figs. 6 and 7, Chloroplast structures of cells exposed to light in the presence of 500 ,ug/ml aminonucleoside. Experimental
Cell Research
47
Aminonucleoside
efects
in Euglena
gracilis
Experimental
243
Cell Research
47
M. I. Selsky
244
[8]. If the AN-mediated inhibition of ribosomal-RNA synthesis involves RNA that is incorporated into the 80 S ribosome type, then the growth rate inhibition shown in Fig. 1 could be attributed to decreased formation of functional cytoplasmic ribosomes. The fact that there is always some cell division after initial AN exposure (Fig. 1) may be due to the cell’s utilization of those cytoplasmic ribosomes already formed. Thus only when cytoplasmic ribosome formation becomes limiting may cell division become arrested. If, on the other hand, one hypothesizes that AN can inhibit synthesis of ribosomal-RNA for both ribosome types, then there is another possible explanation of the results. It may be that there are sufficient ribosomes already present in the proplastids of dark-adapted Euglena which can serve as chloroplast protein synthesis sites during chloroplast development without further chloroplast ribosome formation. Schiff and Epstein [la] have reported that workers in their laboratory have been able to demonstrate the presence of ribosomes in Euglena proplastids by electron microscopy. Thus even if AN did inhibit new chloroplast ribosomal-RNA synthesis, the existing proplastid ribosomes may be sufficient for the programming of many new chloroplast proteins. A third hypothesis to explain the effects observed is that the intact chloroplast or proplastid membrane may be relatively impermeable to AN at low concentrations, and thus shield the chloroplast ribosome from drug effects, However, electron micrographs of cells treated with AN at 500 pg/ml reveal that they possess chloroplasts with abnormal lamellar arrangements or unfused discs. This would indicate that the drug is penetrating the chloroplast or proplastid membrane at high dose levels. Yet, even these cells were shown to accumulate fairly normal chlorophyll levels (Fig. 3) which would indicate that both chlorophyll production and chloroplast protein synthesis are not completely blocked by levels of AN that do permeate the chloroplast or proplastid membrane. The chloroplast ribosomes appear to be relatively refractory to AN-mediated inhibition. It will be recalled that chloroplast protein synthesis can be inhibited in Euglena, but growh rate not affected, by treatment of the cells with chloramphenicol [20]. This has been correlated with the finding that the major site of action for this drug is the 70 S ribosome which is found in the chloroplast [l, 231. By use of AN, however, one can selectively inhibit growth of Euglena while not markedly affecting chlorophyll production or chloroplast development. Whether one can localize this effect to a cytoplasmic ribosome site is still dependent upon presentation of more direct proof of the specific mechanism of action of AN. Experimental
Cell Research
47
Aminonucleoside
effects in Euglena
gracilis
245
SUMMARY
Dark-adapted or light-grown cells of Euglena gracilis show a marked inhibition in growth rate when grown in the light in the presence of the aminonucleoside of puromycin (AN) at concentrations of 50 pg/ml or more. Inhibition of cell division is not immediate, but occurs after several cell doublings. Chlorophyll accumulation in dark-adapted cells placed in the light under growing or non-growing conditions is not markedly affected by AN at doses sufficient to strongly inhibit growth rate (100 ,ug/ml). Electron micrographs of cells treated at this level indicate that normal chloroplast structures are formed. Abnormal chloroplast structures were seen only in cells treated with 500 pg/ml AN, a dose level at which normal chlorophyll/cell amounts accumulate. Several hypotheses are presented to explain the relative insensitivity of the chloroplast or proplastid to AN in the intact Euglena cell. I wish to express my gratitude to Mrs Betty Hershenov for performing the work in this study relating to electron microscopy, and to Miss Carol Patrick for her excellent technical assistance. This work was supported by a grant, CA07062-02, from the United States Public Health Service. REFERENCES ANDERSON, L. A. and SMILLIE, R. M., Biochem. Biophys. Res. Comm. 23, 535 (1966). 2. ARNON, D. I., Plant Physiol. 24, 1 (1949). Y., SCHIFF, J. A. and EPSTEIN, H. T., Plant Physiol. 39, 231 (1964). 3. BEN-SHAUL, G., Biochim. Biophys. Acta 7i, 317 (1963). 4. BRAWERMAN, BRAWERMAN. G. and EISENSTADT, J., Biochim. BioDhus. Aeta 91, 477 (1964). it DICKIE, N., ALEXANDER, C. S. and NAGASAWA, H. T., kiochim. Biophys. Acia 95, 156 (1965). J. and BRAWERMAN, G., Biochim. Biophys. Acta 76, 319 (1963). 7. EISENSTADT, 8. ~ J. Mol. Biol. 10, 392 (1964). 9. FARNHAM, A. E., Virology 27, 73 (1965). 10. FARNHAM, A. E. and DUBIN, D. T., J. Mol. Biol. 14, 55 (1965). 11. GREENBLATT, C. L. and SCHIFF, J. A., J. Protozool. 6, 23 (1959). J., MANDEL, M., EPSTEIN, H. T. and SCHIFF, J. A., Biochem. Biophys. Res. Comm. 13, 12. LEFF, 126 (1963). 13. LEWIS, S. C., SCHIFF, J. A. and EPSTEIN, H. T., J. Protozool. 12, 281 (1965). 14. LYMAN, H., EPSTEIN, H. T. and SCHIFF, J. A., Biochim. Biophys. Acta 50, 301 (1961). 15. NATHANS, D. and NEIDLE, A., Nature 197, 1076 (1963). 16. RABINOVITZ, M., and FISHER, J. M., J. Biol. ‘Chem. 237, 477 (1963). 17. RAY, D. G. and HANAWALT, P. C., J. Mol. Biol. 9, 812 (1964). 18. SCHIFF, J. A. and EPSTEIN, H. T., in M. LOCKE (ed.), Reproduction: Molecular, Subcellular and Cellular. D. 131. Academic Press, New York. 1966. 19. SELSKY, M. I., J. k>otozool. 11 (Suppl.) 26 i1964). ’ 20. SMILLIE, R. M., EVANS, W. R. and LYMAN, H., Brookhaven Sump. Biol. 16, 89 (1964). 21. STERN, A. I., S~HIFF, j. A. and EPSTEIN, fi. Tl, Plant PhysioL 33, 220 (1964). 22. -ibid. 39, 226 (1964). 23. VASQUEZ, D., Symp. Sac. Gen. Microbial. 16, 169 (1966). 1.
Experimental
Cell Research
47
Cell
Research
EFFECTS ON GROWTH
47, 237-245
OF PUROMYCIN AMINONUCLEOSIDE AND CHLOROPLAST DEVELOPMENT EUGLENA GRACILIS M.
Department
237
(1967)
of Biology,
OF
I. SELSKY
Brooklyn
College,
Received
November
Brooklyn,
New
York
11210,
U.S.A.
21. 1966
b!h~r studies of the factors affecting chloroplast development in Euglena gracilis have been made in recent years and a well-documented revielv on this subject has recently appeared [18]. Much evidence has accumulated to suggest that the chloroplast of Euglena with its unique type of DNA4 [5, 12, 171, and its specific type of ribosome (70 S) [4, 71, may indeed function as a self-sufficient unit of biosynthesis within the cell. Smillie et al. [20] demonstrated that under certain conditions chloramphenicol could preferentially inhibit chloroplast protein synthesis of autotrophic cells while not affecting growth rate of heterotrophic Euglena. Vasquez [23] later showed that chloramphenicol preferentially binds to 70 S ribosomes and Anderson and Smillie [l] correlated this finding with the site of its effect in Euglena, the chloroplast ribosome. It was of interest to know, however, if one could affect the cytoplasmic synthesis systems of the cell without markedly affecting the syntheses localized in the chloroplasts. With this in mind we tested the effects of the aminonucleoside of puromycin (AN) on cells of EugZena. A brief account of some of these findings has been presented elsewhere [19]. The effects of AN have been shown by several investigators [6, 9, 15, 161 to be different from those of the parent compound, puromycin. The primary effect of AN is thought to be an inhibition of RNA synthesis, with most marked inhibition of ribosomal-RNA synthesis. In some systems protein synthesis is not directly inhibited. Farnham and Dubin [B] suggest that the effects of AN may be due to its acting as an analogue of uncharged S-RNA, while Dickie et al. [6] hypothesize that AN acts as a false feedback inhibitor of adenine biosynthesis.
MATERIALS
AND
METHODS
Axenic cultures of Euglena gracilis, var. bacillaris, and the Z strain were grown on a medium adjusted to pH 7.4, which had butanol and acetate as its sole carbon Experimental
Cell
Research
47
238
M.
I. Selsky
sources [ll]. Dark-adapted Euglena were obtained by growing cells in light-proof flasks and transferring them several times in a dark room equipped with a green safe-light. Light-grown cells and cells that were developing chloroplasts (greening cells) were maintained under fluorescent lighting with an intensity of about 250 foot-candles. Inocula of late log phase cells (light or dark-grown cells of the bacillaris variety, or light or dark-grown cells of the Z strain) were added to 50 ml erlenmeyer llasks, which contained O-500 /“g/ml of AN (Nutritional Biochemicals Corp.) dissolved in 10 ml of medium. These cells were grown at 26°C in the light with occasionalshaking. At various intervals aliquots were removed for cell counts, total chlorophyll determination and plating. Total cell number was determined by counting in triplicate with a hemocytometer counting chamber in early experiments, and with a Coulter Counter, Model B (Coulter Electronics Corp.) in later experiments. Viability was determined by plating cells [14] after drug treatment on medium without the antibiotic, and incubating the plates in the light. Total chlorophyll was determined by the method of Arnon [2]. EugIena cells of the Z strain used for electron microscope studies were grown in the dark on pH 7.4 medium, washed twice in 0.05 M Tris, pH 7.4, and resuspended in Tris buffer with O-500 pg/ml AN added. These cultures were placed in the light and harvested after four days. The cells were fixed in 1 per cent osmium tetroxide, pH 7.5, buffered with Verona1 acetate (cells treated with 100 and 500 ,ug/ml AN), or in 64 per cent glutaraldehyde and 1 per cent osmic acid (normal, untreated cells). All cells were embedded in Maraglas (Polyscience Co.). Sections of 300-400 A thicknesswere prepared on a Porter-Blum ultra microtome (Model MT2) with a diamond knife, and stained with uranyl acetate and lead. An RCA microscope, Model EMUSG, was used. RESULTS Fig. 1 illustrates the effect of AN on cultures of light-grown and darkadapted cells of the bacillaris variety and the Z strain when grown in the light. The data plotted are from one of three similar experiments performed, and indicate total cell number over a nine day period. It can be seen that AN does not affect growth rate at a dose of 10 rug/ml. However, marked inhibition of growth rate is evident at levels of AN of 100 pg/ml or more in all the cultures. At 50 ,ug/ml, AN effects seem to be intermediate. Growth rate inhibition caused by AN was noted for all four cell types tested, the effect being more severe with greening cells. It should be noted that in all cultures tested division does not cease abruptly upon exposure of the cells to AN, but several cell doublings can occur before the inhibitory effect is observed. At the same time that cell counts were made, samples were plated. Colonies which formed from cells that were treated at 50-100 pg/ml AN appeared later than colonies from untreated cells and were much smaller in size. Experimental
Cell Research
47
ilminonucleoside
effects in Euglena
gracilis
239
Since the plating was done on media without AN, this lag in colony formation indicated that the inhibitory effect of AN on growth was probably reversible. Some loss of viability was noted, however, when cells treated with 200-500 pg/ml AN were plated. All cells from treated cultures that did form colonies formed green colonies. Even though AN markedly inhibited growth rate for all cell types tested, dark-adapted cultures exposed to AN in the light became green. Total chlorophyll/cell levels were determined for such cells after various periods of light exposure. Fig. 2 shows data obtained from an experiment with cells of the bacillaris variety. These cells were forming chloroplasts in the light in the presence of AN dissolved in growth medium. If these data are compared with the pertinent growth data (Fig. 1) it will be seen that there is an
Fig. l.-Effect of aminonucleoside Z-LT, Z-DK refer to light-grown strain, respectively. The numbers
on growth rate of E. gracilis in the light. B-LT, BPDK and and dark-adapted cells of the bacillaris variety and the Z to the right of the curves refer to aminonucleoside level @g/ml). Experimental
Cell
Research
47
240
ill. I. Selsky
accumulation of chlorophyll in cells treated with large amounts of AN just at the time when growth rate levels off and becomes stationary (between days 3 and 5). On the ninth day of light exposure, when a slight increase in cell number occurs, the chlorophyll/cell level drops. This is not unexpected since
Fig. light
2.-Total chlorophyll levels in cells of dark-adapted E. gracilis var. in the presence of aminonucleoside. Drug levels indicated to the right
hacillaris grown of curves.
in the
greater chlorophyll accumulation occurs when growth rate decreases [21]. The untreated cells, which are actively dividing for live days, show a steady rise in chlorophyll level, although never reaching the level of the treated cultures in this experiment. In order to determine the effect of AN on chlorophyll formation and chloroplast development without concomitant cell growth we next tested dark-adapted cells placed in the light under conditions that allowed little if any cell division. Dark-adapted cells were starved in the dark on 0.05 M Tris, pH 7.4 for four days and then resuspended in the buffer plus L4N (O-500 pg/ml). The cells were then placed in the light and samples were taken at intervals. Although cell number was essentially constant under these conditions, it was noted that the amount of total chlorophyll/cell increased in all cultures with time in the light (Table I). No chlorophyll was detected in any cells at “zero” time, but higher levels were found after longer light exposures. Cells treated at 100 pg/ml AN accumulated levels of chlorophyll/ cell similar to untreated cells. Somewhat lower levels of chlorophyll/cell were observed in this experiment for cells treated at 200 pg/ml. Fig. 3, with Experimental
Cell Research
47
Aminonucleoside
effecfs in Euglena
gracilis
241
data from chlorophyll
a different experiment with non-growing cells, illustrates that accumulated even in cells treated at a very high AN level (500
P&4* In order chloroplast
to ascertain structures,
whether electron
chlorophyll microscope
levels were indicative of formed studies were made of cells that
TABLE I. Effect of aminonucleoside on chlorophyll accumulation in nongrowing, dark-adapted Euglena gracilis, var. bacillaris, placed in the 1ight.a AN (wWm1) Hr in light 0 6 24 48 72 96 120 144 168 216
0 Cells/ml 9.8 x lo6 1.1 x 10' 1.13 x 10’ 1.13 x 10’ 1.2 x 10’ 1.1 x 10’ 9.6 x lo6 8.3 x lo6 8.6 x 106 9.5 x 10”
100 Chlor.
b/Cell
0.0 0.33 3.58 8.50 10.46 13.30 14.60 20.10 20.80 21.90
a Figures represent average of three b Expressed as picagrams (pg) total
Cells/ml 9.1 x 106 9.1 x 106 9.0 x 106 9.0 x 106 9.7 x 10s 1.01 x 10’ 1.0 x 10’ 7.2 x lo6 8.2 x log 7.9 x 106
200 Chlor.
b/Cell
0.0 0.42 2.88 9.24 12.60 13.70 13.50 20.60 20.10 23.70
Cells/ml 1.4 1.2 9.6 9.6 8.2 9.3 9.7 7.2 7.2 8.0
x x x x x x x x x x
Chlor.
10’ 10’ 10” lo6 lo6 106 106 lo6 lo6 lo6
b/Ce[l
0.0 0.29 1.46 6.17 10.50 11.20 10.50 16.50 17.90 17.80
determinations. chlorophyll/cell.
greened under non-growing conditions in the presence of AN. Chloroplasts with normal lamellar structures, similar to those observed in untreated cells, mere seen in dark-adapted cells of the Z strain that were exposed to light in the presence of 100 ,ug/ml AN dissolved in Tris buffer, pH 7.4 (Figs. 4 and 5). Cells treated at a very high level of AN (500 pg/ml) possessed abnormal chloroplasts. In some instances these chloroplasts showed many unfused discs (Fig. S), while in other cells the chloroplasts appeared to have incomplete or unusual lamellar systems (Fig. 7). These chloroplasts appeared somewhat similar to those reported by Ben-Shaul et al. [3] for cells grown at 7 foot-candles of light. The 500 pg/ml AN-treated cells differ from the latter, however, for they possess fairly normal chlorophyll/cell levels. It appears that at very high AN levels some stage in the final development of the chloroplast from the proplastid may be inhibited even though chlorophyll accumulation and concomitant early protein synthesis are relatively insensitive to the drug. I6
-
671817
Experimental
Cell Research
47
242
M. I. Selsky
0
10
50
100
200
500
pgm AMINONUCLEOSIDE/ml
Fig. 3.-Total chlorophyll levels in cells of dark-adapted, non-dividing exposed to light in the presence of aminonucleoside. The bar graphs level refer to chlorophyll/cell amounts after 0, 3 and 7 days in the light.
E. gracitis var. bacillaris for each aminonucleoside
DISCUSSION
From the data reported it can be concluded that AN can markedly affect growth rate of Euglena, while not strongly interfering with accumulation of chlorophyll and the development of a chloroplast in a greening cell, That cells accumulate chlorophyll is indicative of the fact that they are synthesizing chloroplast protein [13, 21, 221. Examination of the electron micrographs presented here shows that normal chloroplast lamellar systems are formed in cells treated at levels of AN sufficient to markedly inhibit growth (100 pg/ml). If one accepts the postulate that a specific control mechanism for the synthesis of chloroplast protein exists in Euglena, which Eisenstadt and Brawerman [4, 81 report may be localized on chloroplast ribosomes, then these findings indicate such a control mechanism may be refractory to AN in the intact cell. Since the major effect of AN is believed to be concerned with inhibition of ribosomal-RNA synthesis [lo], one hypothesis presented to explain our observations is that the synthesis of chloroplast ribosomal-RNA may not be greatly affected by AN. Thus the major site of inhibition would be the ribosomal-RNA of the cytoplasmic ribosome. It must be noted, however, that it is still not known if AN affects synthesis of ribosomal-RNA for 70 S, 80 S, or both type of ribosome. Euglena chloroplast ribosomes have been shown to be of the 70 S type, while those of the cytoplasm are of the 80 S variety
Figs. 4-7.-Sections of dark-adapted E. gracitis, Z strain, exposed to light for 4 days in the presence or absence of aminonucleoside dissolved in buffer. Legend: D, disc; L, lamella; OG, osmophilic granule; P, paramylum. Marker indicates 1 p; Fig. 4, Chloroplast structure of cells exposed to light in the absence of aminonucleoside; Fig. 5, Chloroplast structure of cells exposed to light in the presence of 100 pg/ml aminonucleoside; Figs. 6 and 7, Chloroplast structures of cells exposed to light in the presence of 500 ,ug/ml aminonucleoside. Experimental
Cell Research
47
Aminonucleoside
efects
in Euglena
gracilis
Experimental
243
Cell Research
47
M. I. Selsky
244
[8]. If the AN-mediated inhibition of ribosomal-RNA synthesis involves RNA that is incorporated into the 80 S ribosome type, then the growth rate inhibition shown in Fig. 1 could be attributed to decreased formation of functional cytoplasmic ribosomes. The fact that there is always some cell division after initial AN exposure (Fig. 1) may be due to the cell’s utilization of those cytoplasmic ribosomes already formed. Thus only when cytoplasmic ribosome formation becomes limiting may cell division become arrested. If, on the other hand, one hypothesizes that AN can inhibit synthesis of ribosomal-RNA for both ribosome types, then there is another possible explanation of the results. It may be that there are sufficient ribosomes already present in the proplastids of dark-adapted Euglena which can serve as chloroplast protein synthesis sites during chloroplast development without further chloroplast ribosome formation. Schiff and Epstein [la] have reported that workers in their laboratory have been able to demonstrate the presence of ribosomes in Euglena proplastids by electron microscopy. Thus even if AN did inhibit new chloroplast ribosomal-RNA synthesis, the existing proplastid ribosomes may be sufficient for the programming of many new chloroplast proteins. A third hypothesis to explain the effects observed is that the intact chloroplast or proplastid membrane may be relatively impermeable to AN at low concentrations, and thus shield the chloroplast ribosome from drug effects, However, electron micrographs of cells treated with AN at 500 pg/ml reveal that they possess chloroplasts with abnormal lamellar arrangements or unfused discs. This would indicate that the drug is penetrating the chloroplast or proplastid membrane at high dose levels. Yet, even these cells were shown to accumulate fairly normal chlorophyll levels (Fig. 3) which would indicate that both chlorophyll production and chloroplast protein synthesis are not completely blocked by levels of AN that do permeate the chloroplast or proplastid membrane. The chloroplast ribosomes appear to be relatively refractory to AN-mediated inhibition. It will be recalled that chloroplast protein synthesis can be inhibited in Euglena, but growh rate not affected, by treatment of the cells with chloramphenicol [20]. This has been correlated with the finding that the major site of action for this drug is the 70 S ribosome which is found in the chloroplast [l, 231. By use of AN, however, one can selectively inhibit growth of Euglena while not markedly affecting chlorophyll production or chloroplast development. Whether one can localize this effect to a cytoplasmic ribosome site is still dependent upon presentation of more direct proof of the specific mechanism of action of AN. Experimental
Cell Research
47
Aminonucleoside
effects in Euglena
gracilis
245
SUMMARY
Dark-adapted or light-grown cells of Euglena gracilis show a marked inhibition in growth rate when grown in the light in the presence of the aminonucleoside of puromycin (AN) at concentrations of 50 pg/ml or more. Inhibition of cell division is not immediate, but occurs after several cell doublings. Chlorophyll accumulation in dark-adapted cells placed in the light under growing or non-growing conditions is not markedly affected by AN at doses sufficient to strongly inhibit growth rate (100 ,ug/ml). Electron micrographs of cells treated at this level indicate that normal chloroplast structures are formed. Abnormal chloroplast structures were seen only in cells treated with 500 pg/ml AN, a dose level at which normal chlorophyll/cell amounts accumulate. Several hypotheses are presented to explain the relative insensitivity of the chloroplast or proplastid to AN in the intact Euglena cell. I wish to express my gratitude to Mrs Betty Hershenov for performing the work in this study relating to electron microscopy, and to Miss Carol Patrick for her excellent technical assistance. This work was supported by a grant, CA07062-02, from the United States Public Health Service. REFERENCES ANDERSON, L. A. and SMILLIE, R. M., Biochem. Biophys. Res. Comm. 23, 535 (1966). 2. ARNON, D. I., Plant Physiol. 24, 1 (1949). Y., SCHIFF, J. A. and EPSTEIN, H. T., Plant Physiol. 39, 231 (1964). 3. BEN-SHAUL, G., Biochim. Biophys. Acta 7i, 317 (1963). 4. BRAWERMAN, BRAWERMAN. G. and EISENSTADT, J., Biochim. BioDhus. Aeta 91, 477 (1964). it DICKIE, N., ALEXANDER, C. S. and NAGASAWA, H. T., kiochim. Biophys. Acia 95, 156 (1965). J. and BRAWERMAN, G., Biochim. Biophys. Acta 76, 319 (1963). 7. EISENSTADT, 8. ~ J. Mol. Biol. 10, 392 (1964). 9. FARNHAM, A. E., Virology 27, 73 (1965). 10. FARNHAM, A. E. and DUBIN, D. T., J. Mol. Biol. 14, 55 (1965). 11. GREENBLATT, C. L. and SCHIFF, J. A., J. Protozool. 6, 23 (1959). J., MANDEL, M., EPSTEIN, H. T. and SCHIFF, J. A., Biochem. Biophys. Res. Comm. 13, 12. LEFF, 126 (1963). 13. LEWIS, S. C., SCHIFF, J. A. and EPSTEIN, H. T., J. Protozool. 12, 281 (1965). 14. LYMAN, H., EPSTEIN, H. T. and SCHIFF, J. A., Biochim. Biophys. Acta 50, 301 (1961). 15. NATHANS, D. and NEIDLE, A., Nature 197, 1076 (1963). 16. RABINOVITZ, M., and FISHER, J. M., J. Biol. ‘Chem. 237, 477 (1963). 17. RAY, D. G. and HANAWALT, P. C., J. Mol. Biol. 9, 812 (1964). 18. SCHIFF, J. A. and EPSTEIN, H. T., in M. LOCKE (ed.), Reproduction: Molecular, Subcellular and Cellular. D. 131. Academic Press, New York. 1966. 19. SELSKY, M. I., J. k>otozool. 11 (Suppl.) 26 i1964). ’ 20. SMILLIE, R. M., EVANS, W. R. and LYMAN, H., Brookhaven Sump. Biol. 16, 89 (1964). 21. STERN, A. I., S~HIFF, j. A. and EPSTEIN, fi. Tl, Plant PhysioL 33, 220 (1964). 22. -ibid. 39, 226 (1964). 23. VASQUEZ, D., Symp. Sac. Gen. Microbial. 16, 169 (1966). 1.
Experimental
Cell Research
47