- Email: [email protected]
Effect of actinomycin and puromycin on the deoxyribonuclease activity in P. Lividus embryos at various stages of development
Printed in Sweden Copyright Q 1973 by Amdemic Press, Inc. All rights of veproducfion in any form resertw-d
Experimental Cell Research 82 (1973) 351-356
EFFECT OF ACTINOMYCIN DEOXYRIBONUCLEASE AT VARIOUS
AND PUROMYCIN
ACTIVITY
of Molecular
EMBRYOS
STAGES OF DEVELOPMENT
B. DE PETRUCELLIS Laboratory
IN P. LIVIDUS
ON THE
Embryology,
and E. PARIS1 CNR, Arco Felice, Naples, 1tal.v
SUMMARY The pattern of DNAse activity in sea urchin Paracentrotus lividus during early embryonic development is altered by actinomycin. When the drug is added to the embryos soon after fertilization, the decrease of DNAse activity that normally occurs before the onset of gastrulation is prevented. If actinomycin is added when DNAse activity starts to decrease, the enzyme pattern remains the same as in the control. Addition of the drug at late gastrula stage, on the other hand, brings about a transient increase of activity with respect to that of untreated embryos. Puromycin has no effect on DNAse activity during the period from fertilization to the blastula stage, whereas it inhibits the increase of activity which occurs after gastrulation. The type of regulatory mechanism involved is discussed.
We have previously shown that the pattern of DNAse activity changes in a characteristic manner during the development of the sea urchin, Paracentrotus Zividus [l]. During the first 12 h, i.e. from fertilization to the hatching blastula, the enzyme activity remains essentially constant; it then undergoes a sudden dramatic drop which is followed by a rapid rise starting at the onset of gastrulation. It should be noted that the transient decline of the enzyme activity covers the entire blastula stage, the time when cell divisions also drop almost to zero. Moreover, we have shown that two electrophoretically distinct DNAse components can be recognized in homogenates of sea urchin eggs and embryos. Both of these forms have been found in extracts of nuclei and mitochondria [2, 31. Since the fluctuations of DNAse activity in the course of development cannot be accounted for by 24-731808
the presence of inhibitors or activators of the enzyme and, on the other hand, the same two components are present at all developmental stages [2], the possibility of regulatory mechanism operating either at the transcriptional or translational level, or at the level of enzyme degradation, appeared of interest. We report here on the results of experiments aimed at testing these possibilities through the effects of actinomycin and puromycin on the patterns of enzyme activity during development.
MATERIALS AND METHODS lividus were obtained from the Zooloeical Station, Naples. Most experiments were p&formed at the Station Zooloaiaue de Villefranche-surMer and others were done-at the University of Palermo. Paracentrotus
Exptl Cell Res 82 (1973)
352 B. De Petrocellis & E. Parisi
.-*
---____--------
I
I
10
20
10
20
Fig. I. Abscissa: time after the addition of the drug (hours); ordinate: incorporation of the labeled precursors into acid-insoluble material, expressed as % of the control. Inhibitor added: O-O, soon after fertilization; O-0, at late gastrula stages. Inhibition of RNA synthesis by (left) actinomycin C; (right) protein synthesis by puromycin. The inhibitors were added to 75 ml of embryo suspension at a final cont. of 30 pg/ml for actinomycin and 10 pug/ml for puromycin. The samples used to measure the inhibition of RNA synthesis received 50 ,&i of SH-5-uridine (26 mCi/mmole) while those used to measure the inhibition of protein synthesis received 2.5 ,uCi of 14C-L-leucine (312 mCi/ mmole). To compensate the difference in rate of uptake, the samples in which puromycin was added soon after fertilization received in addition 1.5 pmoles of unlabeled leucine while those in which the drug was added at the late gastrula stage received lOpmoles of unlabeled leucine. Incorporation was measured as described in Methods. Chemicals. All chemicals were analytical reagent grade. Salmon sperm DNA was purchased from Sigma. Actinomycin C was gift of Bayer Wuppertal Elberfeld. Puromycin was purchased from Nutritional Biochemical Corporation, Cleveland, Ohio. *H-5Uridine was from Schwarz Bioresearch, Inc., Orangeburg, N.Y. The solubilizer NCS was from Amersham/ Searle Co., Ill.
and washed 3 times with 5 ml of cold 5 % TCA and once with 5 ml of absolute ethanolether (3:l). The samples were then dissolved in 0.5 ml of NCS and counted in a liquid scintillation counter using toluene + liquiflor as scintillation fluid.
Embryo culture and enzyme Ussdy. The embryos were grown at 18-20°C in Millipore-filtered sea water (approx. 5 x lo8 embryos/l) with the drugs (when added) at the indicated concentrations. At various stages of development, the embryos were collected by centrifugation and washed once with 0.63 M NaCl. They were then suspended in 0.025 M Tris-HCl (pH 8) and disrupted by sonication. The nucleolytic activity was tested and measured as described [l]. Protein determinations were carried out by the procedure of Lowry et al. [4].
In order to study the effect of actinomycin and puromycin on the activity level of DNAse, the drugs were added at different times of development, i.e., soon after fertilization, at the blastula stage, and after gastrulation. In this way, we attempted to avoid artefacts arising from a too lengthy exposure of the embryos to the drugs.
Incorporation of labeled precursors into RNA and protein. 3H-5-Uridine and “C-L-leucine were added to the embryo suspensions at the same time as the drugs. At various intervals after the addition of the drug, duplicate 5 ml samples of the suspersion were withdrawn and the embryos collected by centrifugation. A mixture (5 ml) of absolute ethanol and glacial acetic acid (3:l) were added to each sample. The samples were left at 4°C overnight, then centrifuged Exptl Cell Res 82 (1973)
RESULTS
(a) Effect of actinomycin C on DNAse activity
The experiments reported were carried out with an actinomycin C cont. of 30 pg/ml. Previous experiments [5] had established
Regulation of DNAse in sea urchin embryos 353 lO(
1 i
b
1
10
20
30
40
0
10
20
Fig. 2. Abscissa: duration of development (hours) (a); time after actinomycin C addition (hours) (b); ordinate: DNAse activity (units/mg protein). O-O, Control: actinomycin added O-O, soon, A-A, 15 h, W-R, 27 h after fertilizatibn. -
that with this drug concentration the embryos do not develop beyond the blastula stage; during the period from fertilization to blastula, actinomycin C affects neither the rate of cell proliferation nor the incorporation of labeled amino acids into proteins. Its effects are therefore perfectly superimposable to those of actinomycin D. The inhibition of RNA synthesis, as measured by the incorporation of a labeled precursor into acidprecipitable material, was about 70% 10 h after the addition of the drug (fig. la), the extent of inhibition was the same irrespective whether actinomycin C was added soon after fertilization or at the late gastrula stage. The effect of the actinomycin C on the DNAse activity is illustrated in fig. 2a. This shows that when the drug was added soon after fertilization, the level of DNAse activity during the first period until the early blastula stage was the same as in the controls. How-
ever, the decrease of enzyme activity that normally occurs between hatching and the onset of gastrulation was completely suppressed. When actinomycin C was added at the time the enzyme activity started to decrease, the pattern of DNAse activity followed closely that of the controls. Finally, when actinomycin was added 26 h after fertilization, at the early prism stage, an increase of the enzyme activity was observed: indeed, within 7 to 10 h of the addition of the drug, an activity level twice that of controls was attained. The increase was, however, transient and was soon followed by a drop of the enzyme activity. This effect is shown in fig. 2b which, in addition, illustrates the fluctuations of the DNAse activity observed in the normal embryos between the gastrula and the pluteus stages. Similar fluctuations have been observed in several experiments. Exptl Cell Res 82 (1973)
354 B. De Petrocellis & E. Parisi
during the first 8 h, i.e., until the controls had reached the early blastula stage (fig. 3a). On the other hand, when tre treatment was started at the late gastrula stage (fig. 3b), within 4 h after the addition of the drug, the DNAse activity dropped to values well below those of the controls.
+p.
lo-
h
DISCUSSION
The understanding of the mechanism regmating the changes of the level of any given cell protein requires knowledge of the rate of mRNA transcription and translation as well as of the rate of the degradation of the protein. The difficulties involved in the evaluation of these parameters in eukaryotic cells can be partially circumvented by the use of drugs interfering with transcription and translation. There is a large body of evidence, mostly derived from experiments with actinomycin I I I (see for example [6, 7, 81) that strongly sug30 20 0 10 gests that in the sea urchin embryo developFig. 3. Abscissa: time after puromycin addition ment to the blastula stage is largely (though (hours); ordinate: DNAse activity (units/mg protein). not entirely) under control of maternal O-O, Control; O-O, puromycin-treated embryos. Effect of puromycin on DNAse activity in devel- mRNAs; in order for development to prooping P. lividus embryos. Puromycin was added (a) soon; (b) 24 h after fertilization. DNAse activity was ceed through gastrulation embryonic mRNAs measured as previously described [l]. are, however, required. Our data show that between fertilization and the blastula stage the level of enzyme (b) Effect of Puromycin activity is affected neither by actinomycin on DNAse activity nor by puromycin. This may suggesta pool of Puromycin at a cont. of 10 pg/ml results, enzyme molecules present in the unfertilized after 10 h, in a 6CL80‘X0inhibition of protein egg (i.e. synthesized during oogenesis) which synthesis (see fig. 1b). The development of are being used during the early part of development, up to the blastula stage. However, the embryos stopped soon after the addition of the drug, but the embryos did not show an alternative interpretation could be that any visible sign of cytolysis for several hours. puromycin interferes with the synthesis as Ciliary movement was unaffected in gastrulae, well with the degradation of the enzyme. but invagination of endoderm did not pro- Hence, short of precise information about the enzyme turnover the interpretation of these ceed any further. data is bound to be tentative. The conclusion Puromycin added soon after fertilization did not affect the level of DNAse activity the experiments do allow is that during this 2
Exptl Cell Res 82 (1973)
4
6
8
Regulation of DNAse in sea urchin embryos
period of development the enzyme level is not controlled at the transcriptional level. The observation that DNase activity drops during the blastula stage and the drop is prevented by actinomycin provided the drug is administered before the onset of the decline of the enzyme activity may lend itself at least to two alternative interpretations. One interpretation calls for the operation of an inhibitor synthesized in the early blastula stage; this, however, is contradicted by our previous experiments [l]. As already suggested [I] the drop in DNAse activity may be due to degradative processes resulting from synthesis of proteolytic enzymes directed by new, i.e., embryonic mRNA. Actinomycin would thus prevent the degradative processes by interfering with transcription of specific mRNA. The fact that in order for actinomycin to be effective the treatment must precede the onset of the decline of the DNAse activity suggests that transcription is already completed in the early blastula. The experiments further indicate that at the onset of gastrulation active enzyme synthesis begins: indeed the increase of the DNase activity is blocked by puromycin. What is more difficult to interpret is the ‘paradoxical effect’ that actinomycin given after the gastrula stage brings about an increase of the enzyme activity to values higher than those of the controls. A similar effect of actinomycin has been reported earlier in the case of the dCMP-aminohydrolase in the sea urchin embryo [9] while enzyme induction by actinomycin has been described in a variety of other systems [IO-201. One of the best studied casesis that of the regulation of tyrosine aminotransferase (TAT) in a line of rat hepatoma cells. In this case, the ‘paradoxical effect’ [21] has been interpreted as being due to a labile repressor coded for by a short-lived mRNA. It has
355
been postulated that the control of TAT activity is achieved by such a repressor acting at the post-transcriptional level [22]. Application of a similar model to the effect of actinomycin on the DNase activity in the sea urchin embryo depends again on knowledge about the turnover of the enzyme. Indeed, the possibility that regulation of DNase activity occurs at the level of its degradation rather than of its synthesis cannot be ruled out. The observations on the effect of actinomycin on the enzyme activity may, in fact, be compatible with this interpretation. This would also apply to the fluctuations of DNase activity detectable when the observations are made at short intervals between gastrula and pluteus. It is a pleasure to thank Dr A. Monroy and Dr Cl. Tomkins for helpful discussions and critical reading of the manuscript, and Dr Lenchs and Dr SchmidtKastner of Bayer Wuppertal-Elberfeld for the generour gift of actinomycinC. The authors are verygrateful to Dr J. M. Peres, director of the Station Zoologique, Villefranche-sur-Mer and to Dr R. Lallier for their very kind cooperation in supplying the biological material and working facilities.
REFERENCES 1. De Petrocellis, B & Parisi, E, Exptl cell res 73 (1972) 496. 2. 1 Ibid 79 (1973) 53. 3. Parisi, E & De Petrocellis, B, Biochem biophys res commun 49 (1972) 706. 4. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 5. De Petrocellis, B, Tocco, G & Scarano, E. Unpublished data. 6. Gross, P R & Cousineau, G H, Biochem biophys res commun 10 (1963) 321. 7. Gross, P R, Malkin,.L J & Moyer, W A, Proc natl acad sci US 51 (1964) 404. 8. Giudice, G, Mutolo, V & Donatuti, G, Wilhelm Roux archiv 161 Entwicklungsmech Organ (1968)
118.
9. Scarano, E, De Petrocellis, B & Augusti-Tocco. G, Biochim biophys acta 87 (1964) 174. 10. Nitowsky, H, Geller, S & Casper, R, Fed proc 23 (1964) 556.
11. Kapp, L N & Okada, S, Exptl cell res 72 ( 1972) 473. 12. McAuslan, B R, Virology 21 (1963) 383.
13. Hilf, R, Michel, J, Silverstein, G &Bell, C, Cancer res 25 (1965)
1854.
Exptl Cell Res 82 (1973)
356 B. De Petrocellis & E. Parisi 14. Alescio, T, Moscona, M & Moscona, A A, Exptl cell res 61 (1970) 342. 15. Moscona, A A, Moscona, M H & Saenz, N, Proc natl acad sci US 61 (1968) 160. 16. Moscona, A A, Moscona, M H & Jones, R E, Biochem biophys res commun 39 (1970) 943. 17. Weissman, H & Ben-Oz, S, Biochem biophys res commun 41 (1970) 260. 18. Coleman, G & Elliot, W H, Biochem j 95 (1965)
20. Della Corte, E & Stirpe, F, Biochem j 102 (1967) 520.
21. Garren, L D, Howell, R R, Tomkins, G M & Crocco, R M, Proc natl acad sci US 52 (1964) 1121. 22. Tomkins, G M, Gelehrter, T D, Granner, D, Martin, D jr, Samuels, H H & Thompson, E B, Science 166 (1969) 1474.
699.
19. Horowitz, N H, Feldman, H M & Pall, M L, J biol them 245 (1970) 2784.
Exptl Cell Res 82 (1973)
Received March 20, 1973 Revised version received June 25, 1973
Experimental Cell Research 82 (1973) 351-356
EFFECT OF ACTINOMYCIN DEOXYRIBONUCLEASE AT VARIOUS
AND PUROMYCIN
ACTIVITY
of Molecular
EMBRYOS
STAGES OF DEVELOPMENT
B. DE PETRUCELLIS Laboratory
IN P. LIVIDUS
ON THE
Embryology,
and E. PARIS1 CNR, Arco Felice, Naples, 1tal.v
SUMMARY The pattern of DNAse activity in sea urchin Paracentrotus lividus during early embryonic development is altered by actinomycin. When the drug is added to the embryos soon after fertilization, the decrease of DNAse activity that normally occurs before the onset of gastrulation is prevented. If actinomycin is added when DNAse activity starts to decrease, the enzyme pattern remains the same as in the control. Addition of the drug at late gastrula stage, on the other hand, brings about a transient increase of activity with respect to that of untreated embryos. Puromycin has no effect on DNAse activity during the period from fertilization to the blastula stage, whereas it inhibits the increase of activity which occurs after gastrulation. The type of regulatory mechanism involved is discussed.
We have previously shown that the pattern of DNAse activity changes in a characteristic manner during the development of the sea urchin, Paracentrotus Zividus [l]. During the first 12 h, i.e. from fertilization to the hatching blastula, the enzyme activity remains essentially constant; it then undergoes a sudden dramatic drop which is followed by a rapid rise starting at the onset of gastrulation. It should be noted that the transient decline of the enzyme activity covers the entire blastula stage, the time when cell divisions also drop almost to zero. Moreover, we have shown that two electrophoretically distinct DNAse components can be recognized in homogenates of sea urchin eggs and embryos. Both of these forms have been found in extracts of nuclei and mitochondria [2, 31. Since the fluctuations of DNAse activity in the course of development cannot be accounted for by 24-731808
the presence of inhibitors or activators of the enzyme and, on the other hand, the same two components are present at all developmental stages [2], the possibility of regulatory mechanism operating either at the transcriptional or translational level, or at the level of enzyme degradation, appeared of interest. We report here on the results of experiments aimed at testing these possibilities through the effects of actinomycin and puromycin on the patterns of enzyme activity during development.
MATERIALS AND METHODS lividus were obtained from the Zooloeical Station, Naples. Most experiments were p&formed at the Station Zooloaiaue de Villefranche-surMer and others were done-at the University of Palermo. Paracentrotus
Exptl Cell Res 82 (1973)
352 B. De Petrocellis & E. Parisi
.-*
---____--------
I
I
10
20
10
20
Fig. I. Abscissa: time after the addition of the drug (hours); ordinate: incorporation of the labeled precursors into acid-insoluble material, expressed as % of the control. Inhibitor added: O-O, soon after fertilization; O-0, at late gastrula stages. Inhibition of RNA synthesis by (left) actinomycin C; (right) protein synthesis by puromycin. The inhibitors were added to 75 ml of embryo suspension at a final cont. of 30 pg/ml for actinomycin and 10 pug/ml for puromycin. The samples used to measure the inhibition of RNA synthesis received 50 ,&i of SH-5-uridine (26 mCi/mmole) while those used to measure the inhibition of protein synthesis received 2.5 ,uCi of 14C-L-leucine (312 mCi/ mmole). To compensate the difference in rate of uptake, the samples in which puromycin was added soon after fertilization received in addition 1.5 pmoles of unlabeled leucine while those in which the drug was added at the late gastrula stage received lOpmoles of unlabeled leucine. Incorporation was measured as described in Methods. Chemicals. All chemicals were analytical reagent grade. Salmon sperm DNA was purchased from Sigma. Actinomycin C was gift of Bayer Wuppertal Elberfeld. Puromycin was purchased from Nutritional Biochemical Corporation, Cleveland, Ohio. *H-5Uridine was from Schwarz Bioresearch, Inc., Orangeburg, N.Y. The solubilizer NCS was from Amersham/ Searle Co., Ill.
and washed 3 times with 5 ml of cold 5 % TCA and once with 5 ml of absolute ethanolether (3:l). The samples were then dissolved in 0.5 ml of NCS and counted in a liquid scintillation counter using toluene + liquiflor as scintillation fluid.
Embryo culture and enzyme Ussdy. The embryos were grown at 18-20°C in Millipore-filtered sea water (approx. 5 x lo8 embryos/l) with the drugs (when added) at the indicated concentrations. At various stages of development, the embryos were collected by centrifugation and washed once with 0.63 M NaCl. They were then suspended in 0.025 M Tris-HCl (pH 8) and disrupted by sonication. The nucleolytic activity was tested and measured as described [l]. Protein determinations were carried out by the procedure of Lowry et al. [4].
In order to study the effect of actinomycin and puromycin on the activity level of DNAse, the drugs were added at different times of development, i.e., soon after fertilization, at the blastula stage, and after gastrulation. In this way, we attempted to avoid artefacts arising from a too lengthy exposure of the embryos to the drugs.
Incorporation of labeled precursors into RNA and protein. 3H-5-Uridine and “C-L-leucine were added to the embryo suspensions at the same time as the drugs. At various intervals after the addition of the drug, duplicate 5 ml samples of the suspersion were withdrawn and the embryos collected by centrifugation. A mixture (5 ml) of absolute ethanol and glacial acetic acid (3:l) were added to each sample. The samples were left at 4°C overnight, then centrifuged Exptl Cell Res 82 (1973)
RESULTS
(a) Effect of actinomycin C on DNAse activity
The experiments reported were carried out with an actinomycin C cont. of 30 pg/ml. Previous experiments [5] had established
Regulation of DNAse in sea urchin embryos 353 lO(
1 i
b
1
10
20
30
40
0
10
20
Fig. 2. Abscissa: duration of development (hours) (a); time after actinomycin C addition (hours) (b); ordinate: DNAse activity (units/mg protein). O-O, Control: actinomycin added O-O, soon, A-A, 15 h, W-R, 27 h after fertilizatibn. -
that with this drug concentration the embryos do not develop beyond the blastula stage; during the period from fertilization to blastula, actinomycin C affects neither the rate of cell proliferation nor the incorporation of labeled amino acids into proteins. Its effects are therefore perfectly superimposable to those of actinomycin D. The inhibition of RNA synthesis, as measured by the incorporation of a labeled precursor into acidprecipitable material, was about 70% 10 h after the addition of the drug (fig. la), the extent of inhibition was the same irrespective whether actinomycin C was added soon after fertilization or at the late gastrula stage. The effect of the actinomycin C on the DNAse activity is illustrated in fig. 2a. This shows that when the drug was added soon after fertilization, the level of DNAse activity during the first period until the early blastula stage was the same as in the controls. How-
ever, the decrease of enzyme activity that normally occurs between hatching and the onset of gastrulation was completely suppressed. When actinomycin C was added at the time the enzyme activity started to decrease, the pattern of DNAse activity followed closely that of the controls. Finally, when actinomycin was added 26 h after fertilization, at the early prism stage, an increase of the enzyme activity was observed: indeed, within 7 to 10 h of the addition of the drug, an activity level twice that of controls was attained. The increase was, however, transient and was soon followed by a drop of the enzyme activity. This effect is shown in fig. 2b which, in addition, illustrates the fluctuations of the DNAse activity observed in the normal embryos between the gastrula and the pluteus stages. Similar fluctuations have been observed in several experiments. Exptl Cell Res 82 (1973)
354 B. De Petrocellis & E. Parisi
during the first 8 h, i.e., until the controls had reached the early blastula stage (fig. 3a). On the other hand, when tre treatment was started at the late gastrula stage (fig. 3b), within 4 h after the addition of the drug, the DNAse activity dropped to values well below those of the controls.
+p.
lo-
h
DISCUSSION
The understanding of the mechanism regmating the changes of the level of any given cell protein requires knowledge of the rate of mRNA transcription and translation as well as of the rate of the degradation of the protein. The difficulties involved in the evaluation of these parameters in eukaryotic cells can be partially circumvented by the use of drugs interfering with transcription and translation. There is a large body of evidence, mostly derived from experiments with actinomycin I I I (see for example [6, 7, 81) that strongly sug30 20 0 10 gests that in the sea urchin embryo developFig. 3. Abscissa: time after puromycin addition ment to the blastula stage is largely (though (hours); ordinate: DNAse activity (units/mg protein). not entirely) under control of maternal O-O, Control; O-O, puromycin-treated embryos. Effect of puromycin on DNAse activity in devel- mRNAs; in order for development to prooping P. lividus embryos. Puromycin was added (a) soon; (b) 24 h after fertilization. DNAse activity was ceed through gastrulation embryonic mRNAs measured as previously described [l]. are, however, required. Our data show that between fertilization and the blastula stage the level of enzyme (b) Effect of Puromycin activity is affected neither by actinomycin on DNAse activity nor by puromycin. This may suggesta pool of Puromycin at a cont. of 10 pg/ml results, enzyme molecules present in the unfertilized after 10 h, in a 6CL80‘X0inhibition of protein egg (i.e. synthesized during oogenesis) which synthesis (see fig. 1b). The development of are being used during the early part of development, up to the blastula stage. However, the embryos stopped soon after the addition of the drug, but the embryos did not show an alternative interpretation could be that any visible sign of cytolysis for several hours. puromycin interferes with the synthesis as Ciliary movement was unaffected in gastrulae, well with the degradation of the enzyme. but invagination of endoderm did not pro- Hence, short of precise information about the enzyme turnover the interpretation of these ceed any further. data is bound to be tentative. The conclusion Puromycin added soon after fertilization did not affect the level of DNAse activity the experiments do allow is that during this 2
Exptl Cell Res 82 (1973)
4
6
8
Regulation of DNAse in sea urchin embryos
period of development the enzyme level is not controlled at the transcriptional level. The observation that DNase activity drops during the blastula stage and the drop is prevented by actinomycin provided the drug is administered before the onset of the decline of the enzyme activity may lend itself at least to two alternative interpretations. One interpretation calls for the operation of an inhibitor synthesized in the early blastula stage; this, however, is contradicted by our previous experiments [l]. As already suggested [I] the drop in DNAse activity may be due to degradative processes resulting from synthesis of proteolytic enzymes directed by new, i.e., embryonic mRNA. Actinomycin would thus prevent the degradative processes by interfering with transcription of specific mRNA. The fact that in order for actinomycin to be effective the treatment must precede the onset of the decline of the DNAse activity suggests that transcription is already completed in the early blastula. The experiments further indicate that at the onset of gastrulation active enzyme synthesis begins: indeed the increase of the DNase activity is blocked by puromycin. What is more difficult to interpret is the ‘paradoxical effect’ that actinomycin given after the gastrula stage brings about an increase of the enzyme activity to values higher than those of the controls. A similar effect of actinomycin has been reported earlier in the case of the dCMP-aminohydrolase in the sea urchin embryo [9] while enzyme induction by actinomycin has been described in a variety of other systems [IO-201. One of the best studied casesis that of the regulation of tyrosine aminotransferase (TAT) in a line of rat hepatoma cells. In this case, the ‘paradoxical effect’ [21] has been interpreted as being due to a labile repressor coded for by a short-lived mRNA. It has
355
been postulated that the control of TAT activity is achieved by such a repressor acting at the post-transcriptional level [22]. Application of a similar model to the effect of actinomycin on the DNase activity in the sea urchin embryo depends again on knowledge about the turnover of the enzyme. Indeed, the possibility that regulation of DNase activity occurs at the level of its degradation rather than of its synthesis cannot be ruled out. The observations on the effect of actinomycin on the enzyme activity may, in fact, be compatible with this interpretation. This would also apply to the fluctuations of DNase activity detectable when the observations are made at short intervals between gastrula and pluteus. It is a pleasure to thank Dr A. Monroy and Dr Cl. Tomkins for helpful discussions and critical reading of the manuscript, and Dr Lenchs and Dr SchmidtKastner of Bayer Wuppertal-Elberfeld for the generour gift of actinomycinC. The authors are verygrateful to Dr J. M. Peres, director of the Station Zoologique, Villefranche-sur-Mer and to Dr R. Lallier for their very kind cooperation in supplying the biological material and working facilities.
REFERENCES 1. De Petrocellis, B & Parisi, E, Exptl cell res 73 (1972) 496. 2. 1 Ibid 79 (1973) 53. 3. Parisi, E & De Petrocellis, B, Biochem biophys res commun 49 (1972) 706. 4. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 5. De Petrocellis, B, Tocco, G & Scarano, E. Unpublished data. 6. Gross, P R & Cousineau, G H, Biochem biophys res commun 10 (1963) 321. 7. Gross, P R, Malkin,.L J & Moyer, W A, Proc natl acad sci US 51 (1964) 404. 8. Giudice, G, Mutolo, V & Donatuti, G, Wilhelm Roux archiv 161 Entwicklungsmech Organ (1968)
118.
9. Scarano, E, De Petrocellis, B & Augusti-Tocco. G, Biochim biophys acta 87 (1964) 174. 10. Nitowsky, H, Geller, S & Casper, R, Fed proc 23 (1964) 556.
11. Kapp, L N & Okada, S, Exptl cell res 72 ( 1972) 473. 12. McAuslan, B R, Virology 21 (1963) 383.
13. Hilf, R, Michel, J, Silverstein, G &Bell, C, Cancer res 25 (1965)
1854.
Exptl Cell Res 82 (1973)
356 B. De Petrocellis & E. Parisi 14. Alescio, T, Moscona, M & Moscona, A A, Exptl cell res 61 (1970) 342. 15. Moscona, A A, Moscona, M H & Saenz, N, Proc natl acad sci US 61 (1968) 160. 16. Moscona, A A, Moscona, M H & Jones, R E, Biochem biophys res commun 39 (1970) 943. 17. Weissman, H & Ben-Oz, S, Biochem biophys res commun 41 (1970) 260. 18. Coleman, G & Elliot, W H, Biochem j 95 (1965)
20. Della Corte, E & Stirpe, F, Biochem j 102 (1967) 520.
21. Garren, L D, Howell, R R, Tomkins, G M & Crocco, R M, Proc natl acad sci US 52 (1964) 1121. 22. Tomkins, G M, Gelehrter, T D, Granner, D, Martin, D jr, Samuels, H H & Thompson, E B, Science 166 (1969) 1474.
699.
19. Horowitz, N H, Feldman, H M & Pall, M L, J biol them 245 (1970) 2784.
Exptl Cell Res 82 (1973)
Received March 20, 1973 Revised version received June 25, 1973