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Nucleic acid and protein metabolism of the Ilyanassa embryo
320
Cell Research 24, 320-326 (IQGI)
Experimental
NUCLEIC
ACID AND PROTEIN METABOLISM THE ILYANASSA EMBRYO1
OF
J. R. COLLIER Marine
Biological
Laboratory,
Woods Hole, Massachusetts,
U.S.A.
Received December 19, 1960
PREVIOLJS experiments [3] with the Ilyanassa egg and subsequent determinations at later stages showed that, with the exception of minor fluctuations, no synthesis of ribonucleic acid (RNA) occurred until the fourth day of development. These results raised the question as to whether the RNA of the egg was functional as judged by metabolic activity during the early stages of development. In the experiments reported in this paper a comparison is made between the time of synthesis of new RNA and the incorporation of 32P into the RNA of the Ilyanassa embryo. Some data on the incorporation of 14C leucine into the proteins are also compared to the metabolism of RNA.
MATERIALS
AND
METHODS
Eggs were obtained from snails kept in an aquarium of running sea water in which usually an abundance of egg capsules were deposited daily. Eggs were removed from their capsules and washed several times in pasteurized sea water before use in each experiment. All embryos were reared at 18°C. Embryos of each stage were incubated for three hours at room temperature (22°C) in sea water containing 1 microcurie of aaP as orthophosphate per milliliter. Before further treatment the embryos were removed from the radioactive phosphorus and washed in several changes of sea water. A sample of the final wash water was counted and found to be at background level. The RNA was extracted from the embryos exposed to 32P according to the procedure of Ogur and Rosen [ll]; 100 embryos were used in each determination. The details of the procedure and its effectiveness with this material in separating the RNA from the cellular milieu have been described and critically discussed previously [3]. Quantitation of the RNA was achieved by ultraviolet spectrophotometry as described earlier [Sl. The RNA hydrolyzate, contained in 50 microliters of 1 N perchloric acid, was planchetted and counted in a gas flow counter with an ultrathin window. 1 This work was supported by a research Metabolic Diseases of the National Institutes Experimental
Cell Research 24
grant (A-3554) of Health.
from the Division
of Arthritis
and
RNA synthesis in the Ilyanassa embryo
321
For the incorporation studies of leucine into the proteins twenty-five embryos were incubated for three hours at room temperature in sea water containing 0.5 microcurie of DL-leucine-1-W. After incubation with the labelled leucine the embryos were washed in several changes of sea water and then homogenized in a small volume of sea water. The proteins of the homogenate were precipitated by the addition of cold 0.5 1cTperchloric acid. The protein residue was washed with two additional cold 0.5 X perchloric acid washes to remove all acid soluble components. The proteins were further purified by two ethanol-ether extractions to remove the lipids and one 0.5 X perchloric acid extraction at 80°C for 15 minutes to remove the nucleic acids. The protein residue was planchetted and counted as indicated above. Because of the small amount of material on the planchet it was not necessary to correct for sclfabsorption. RESULTS The results for the incorporation of 321’ into the RNA are presented as counts per minute per one hundred embryos, and the data for the synthesis of RN-1 for later stages, which represents the means of at least seven determinations for each stage, are given as the percentage difference from the RX;\ emhr?o takes up difYerent content of the uncleaved egg. Because the Ilyrrnasso amounts of 14C leucine at different stages of development the data for the incorporation of 14Cleucine into the protein fraction are given as the ratio ot counts per minute per ‘25 embryos of the protein fraction IO that of the acid soluble fraction. In Fig. 1 the data for the synthesis of RNA and for 32E)incorporation into the RNA are presented. The small changes in the R9A content of the embryo up to the third day of development will be considered as indicating no net synthesis of RSA during this time interval. JVhile these fluctuations appear to he statistically significant, additional determinations are required hefore their significance can be established. Thus, in Fig. 1 it is seen that the first synthesis of Rh’h occurs during the fourth day and that this perioct of active R?;A synthesis continues throughout the fifth day. .1t later stages the RX;h level remains nearly the same with the exception of a possihlc increase during the seventh and last day of development. .\lso in Fig. 1 are the data for 32P incorporation into the RSA, which she\\- that a sharp increase in 32E’ incorporation occurs during the third dav of development. This first increase in incorporation, which is actually the first stag< showing a substantial incorporation of 32P into the RSi?, occurs ahout Ilventy-four hours before any net synthesis of RSA takes place. !)uring all later stages of development there is a close parallel het\vecn RSA synthesis and incorporation of 32P. The values given for the incorporation of phos-
322
J. R. Collier
phorus during the fifth day, while of the correct order of magnitude, may be slightly high, because the data for this stage were obtained from a different experiment; otherwise, the data for all other stages were obtained from the same experiment, in which all embryos were incubated and fractionated at the same time. 10,000 =
-280
6000:
,I- 260
4000 1 2000; lOOO= ?? 2
600:
8 400c .E E ? 2oo$
IOO80 =
i e
601
10-l
-20 0
Fig. l.-RNA content.
;
synthesis
i
i :, Days of development, lEcC
and 32P incorporation.
(1) incorporation
i
i
;
of ‘*P into the RNA,
(2) RNA
In Fig. 2 the incorporation of 14C leucine into the protein fraction is compared with the incorporation of 32P into the RNA, and was found a close correlation in time between these two events during all stages of development. Of particular interest is the fact that the first significant incorporation of 14C leucine into the proteins, presumably indicating protein synthesis, occurs during the third day, at which time no net synthesis of RNA has occurred; however, there is a marked increase in metabolic activity of the RNA as indicated by a substantial incorporation of 32P at this stage. The only significant departure in this correlation between the exchange of RKA phosphorus and amino acid incorporation is that the maximal turnover of Experimental
Cell Research 24
323
l&VA synthesis in the llyanassa embqo RSA phosphorus occurs masimal incorporation earlier. Iluring the first three visible organ primordia 10.000
during the fifth day of development, whereas the of leucine into the protein fraction occurs a day days of development of the flyrrr~rrsstr embryo no are formed as this period is chielly one of cleavage,
1
6000: 4000 1 30002000-
r\
loot-
\
- 1.o aI
"0 lOOO-
-0.9
2 s - 0.8 z : - 0.7 9 - 0.6 .; s -0.5 &
‘0, \
- 0.4 ,j -0.3 -0.2
‘0 -10 1 0
;
;
i
z
Days of development,
Fig. 2.--32P incorporation
into RNA
;
il
c 0 ;
. -0 0.1 g : r 0.0 7
18 C
and leucine-l-l%
incorporation
into protein.
determination and gastrulation. The marked changes in the metabolism of nucleic acids and proteins during the fourth day is not associated with gastrulation as this event is completed hy the end of the second day of development. The onset of RNA synthesis and the increase in incorporation of amino acids found during the fourth day precedes by one day the appearThe final two days of development consist ance of the organ primordia. of further elaboration and growth of the larval organs, and by the end of the seventh day a fully differentiated veliger larva is formed. As shown in Figs. 1 and 2, there is no significant synthesis of RX.4 nor incorporation of amino acids into the proteins during the last t\vo days of development.
J. X. Collier
324
DISCUSSION
The virtual absence of any incorporation of phosphorus into the RNA during the first two days reflects the passive role of this nucleic acid during the early development of the Ilyanassa embryo. Though the segregation of ribonucleoprotein to the various germinal regions during early cleavage may be of utmost importance for normal embryogenesis, the RNA at this stage appears to be inactive. This situation is analogous to that of the deoxyexcept at the time of their synthesis, and is what might be nucleoproteins, expected if the ribonucleoproteins serve as mediators of genetic information to the cytoplasm during development. The discrepancy observed between the time when the RN,4 of the Ilyanassa embryo begins to incorporate radioactive phosphorus and the period when the first synthesis of RNA occurs shows that the incorporation of 32P is an inadequate criterion for detecting the time of RNA synthesis. It is possible that the incorporation of phosphorus into the RNA observed during the third day is related to the apparent decrease in RNA found at that time. At present it is not possible to decide this point because of the uncertainty about the reality of this decrease in the RNA content. Otherwise the incorporation of 32P before any increase in RNA content may indicate that the RNA of the egg has begun to play some functional role in the embryo’s metabolism prior to the time when new RNA is synthesized. The close correlation between the incorporation of phosphorus in the RNA and the incorporation of 14C-leucine into the proteins strongly suggests that this is the case. As pointed out previously, there is relatively little synthetic activity during the early stages of development of the Ilyanassa embryo. In this embryo there is no increase in the incorporation of leucine into the proteins at the time of gastrulation. The small changes observed in the RNA content after the first day of development are of doubtful significance. There is no increase in the incorporation of 32P into the RNA during gastrulation, and this casts some doubt on the validity of the minor fluctuations in the RNA content reported above. As a tentative conclusion it appears that in the Ilyanasscr embryo there is very little synthetic activity at the time of embryonic determination, which occurs during early cleavage. The production of new RNA and proteins, which does not take place until the fourth day of development, begins intenselyjust before the onset of histological differentiation. Cohen [a] and Flickinger [5] found that at gastrulation there is a marked Experimental
Cell Research 24
KNL4 synthesis in the Ilyunassa embryo
325
increase in the incorporation of 14C-labeleci carbon from carbon dioxide into the proteins of the frog embryo. Hultin [9], using a cell-free system from the sea urchin embryo, studied the incorporation of ‘K-1 -1cucino into the proteins and found that incorporation began soon after fertilization anti culminated in the late blastula stage. Earlier, Hultin [lo ( had follo~vetl thtb incwqwration of 14C-lahelctl carbon dioxide into the proteins of the sea and had found that the specific activity of the carhoxyl urchin crnhyo reached a peak shortly before 111~ carbon incrcasetl just after fertilization, nnrwnc~hymc blastula stage, and increased again at the beginning of gastrul:ltion. \‘ille 11‘L 1 has reported on the uptake of 321’ into the RN>4 of the sea urchin embryo. From this data it appears that there is no increase in the incorporw tion 01’ I)hosphorus through the blastula stage, and any increase at the time of gastrulation is doubtful. Hultin [M-4] has studied the contributions 01 1”s labcllctl ammonium chloride, 14C-formate, 14(:-fornlatc, “(I-varbonatc and I*(:-acetate to Ihe RNA1 of the de\-eloping sea uwhin embryo, \vhcrcs tic found an intensified incorporation of labelled atonis from formale, carhonatc anti ammonium chloride during the blastula stage. The lahcllcct carbon from acetate \vas intensively incorporated during the mesrnch~mc~ I)lastula stage. Klson (It rrl. T-1 have sho\vn that an increase in the RX.-\ c~onl~~nl of the sea urchin embryo occur just before gastrulation, and ISSc-I
SUMMARY
The incorporation of 32P into the R?i,I and of nl.-leucinc-1 -14C into the proteins \vas determined at various developmental stages of the Ilytrnrt.sstr embryo. A significant incorporation of 321’ into the KS.4 owurrecl before any net synthesis of HSA.
J. H. Collier
326
A close correlation was found between the incorporation of phosphorus into the RI%A and the incorporation of 14C-leucine into the proteins. There was no increased protein or RNA metabolism associated with gastrulation. The period of intensive synthesis of protein and RNA was found to occur after embryonic determination and just before the beginning of histological differentiation. The author wishes to express his thanks to Marjorie M. Collier for her excellent assistance during the course of this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
B:~cKsTR~&~, S., A&iv 2001. 12, 339 (1959). COHEN, S., J. Biol. Chem., 211, 337 (1954). COLLIER, J. R., Exptl. Cell Research 21, 126 (1960). ELSON. D.. GUSTAFSON. T. and CHARGAFF, E., J. &I[. FLICKI’NGE~, R. A., Ex&l. Cell Research 6; 172 (1954). HULTIN, T., Arkiu Kemi 5, 267 (1953). __ Ibid. 6, 195 (1953).
Chem. 209, 285 (1954).
-__
Exptl. Cell Research 12, 518 (1957). Developmental Biol. 2, 61 (1960). HULTIN, T. grid WESSEL, G., Eiptl. Cell Research 3, 613 (1952). OGUR, hl. and ROSEN, G., Arch. Biochem. 25, 262 (1950).
VILLE,
C. A., LOWENS, M., GORDON, Physiol. 33, 93 (1949).
Experimenlal
Cell Research 24
M.,
LEOXARD,
E.
and
RICH,
A.,
.J. Cellular
Comp.
Cell Research 24, 320-326 (IQGI)
Experimental
NUCLEIC
ACID AND PROTEIN METABOLISM THE ILYANASSA EMBRYO1
OF
J. R. COLLIER Marine
Biological
Laboratory,
Woods Hole, Massachusetts,
U.S.A.
Received December 19, 1960
PREVIOLJS experiments [3] with the Ilyanassa egg and subsequent determinations at later stages showed that, with the exception of minor fluctuations, no synthesis of ribonucleic acid (RNA) occurred until the fourth day of development. These results raised the question as to whether the RNA of the egg was functional as judged by metabolic activity during the early stages of development. In the experiments reported in this paper a comparison is made between the time of synthesis of new RNA and the incorporation of 32P into the RNA of the Ilyanassa embryo. Some data on the incorporation of 14C leucine into the proteins are also compared to the metabolism of RNA.
MATERIALS
AND
METHODS
Eggs were obtained from snails kept in an aquarium of running sea water in which usually an abundance of egg capsules were deposited daily. Eggs were removed from their capsules and washed several times in pasteurized sea water before use in each experiment. All embryos were reared at 18°C. Embryos of each stage were incubated for three hours at room temperature (22°C) in sea water containing 1 microcurie of aaP as orthophosphate per milliliter. Before further treatment the embryos were removed from the radioactive phosphorus and washed in several changes of sea water. A sample of the final wash water was counted and found to be at background level. The RNA was extracted from the embryos exposed to 32P according to the procedure of Ogur and Rosen [ll]; 100 embryos were used in each determination. The details of the procedure and its effectiveness with this material in separating the RNA from the cellular milieu have been described and critically discussed previously [3]. Quantitation of the RNA was achieved by ultraviolet spectrophotometry as described earlier [Sl. The RNA hydrolyzate, contained in 50 microliters of 1 N perchloric acid, was planchetted and counted in a gas flow counter with an ultrathin window. 1 This work was supported by a research Metabolic Diseases of the National Institutes Experimental
Cell Research 24
grant (A-3554) of Health.
from the Division
of Arthritis
and
RNA synthesis in the Ilyanassa embryo
321
For the incorporation studies of leucine into the proteins twenty-five embryos were incubated for three hours at room temperature in sea water containing 0.5 microcurie of DL-leucine-1-W. After incubation with the labelled leucine the embryos were washed in several changes of sea water and then homogenized in a small volume of sea water. The proteins of the homogenate were precipitated by the addition of cold 0.5 1cTperchloric acid. The protein residue was washed with two additional cold 0.5 X perchloric acid washes to remove all acid soluble components. The proteins were further purified by two ethanol-ether extractions to remove the lipids and one 0.5 X perchloric acid extraction at 80°C for 15 minutes to remove the nucleic acids. The protein residue was planchetted and counted as indicated above. Because of the small amount of material on the planchet it was not necessary to correct for sclfabsorption. RESULTS The results for the incorporation of 321’ into the RNA are presented as counts per minute per one hundred embryos, and the data for the synthesis of RN-1 for later stages, which represents the means of at least seven determinations for each stage, are given as the percentage difference from the RX;\ emhr?o takes up difYerent content of the uncleaved egg. Because the Ilyrrnasso amounts of 14C leucine at different stages of development the data for the incorporation of 14Cleucine into the protein fraction are given as the ratio ot counts per minute per ‘25 embryos of the protein fraction IO that of the acid soluble fraction. In Fig. 1 the data for the synthesis of RNA and for 32E)incorporation into the RNA are presented. The small changes in the R9A content of the embryo up to the third day of development will be considered as indicating no net synthesis of RSA during this time interval. JVhile these fluctuations appear to he statistically significant, additional determinations are required hefore their significance can be established. Thus, in Fig. 1 it is seen that the first synthesis of Rh’h occurs during the fourth day and that this perioct of active R?;A synthesis continues throughout the fifth day. .1t later stages the RX;h level remains nearly the same with the exception of a possihlc increase during the seventh and last day of development. .\lso in Fig. 1 are the data for 32P incorporation into the RSA, which she\\- that a sharp increase in 32E’ incorporation occurs during the third dav of development. This first increase in incorporation, which is actually the first stag< showing a substantial incorporation of 32P into the RSi?, occurs ahout Ilventy-four hours before any net synthesis of RSA takes place. !)uring all later stages of development there is a close parallel het\vecn RSA synthesis and incorporation of 32P. The values given for the incorporation of phos-
322
J. R. Collier
phorus during the fifth day, while of the correct order of magnitude, may be slightly high, because the data for this stage were obtained from a different experiment; otherwise, the data for all other stages were obtained from the same experiment, in which all embryos were incubated and fractionated at the same time. 10,000 =
-280
6000:
,I- 260
4000 1 2000; lOOO= ?? 2
600:
8 400c .E E ? 2oo$
IOO80 =
i e
601
10-l
-20 0
Fig. l.-RNA content.
;
synthesis
i
i :, Days of development, lEcC
and 32P incorporation.
(1) incorporation
i
i
;
of ‘*P into the RNA,
(2) RNA
In Fig. 2 the incorporation of 14C leucine into the protein fraction is compared with the incorporation of 32P into the RNA, and was found a close correlation in time between these two events during all stages of development. Of particular interest is the fact that the first significant incorporation of 14C leucine into the proteins, presumably indicating protein synthesis, occurs during the third day, at which time no net synthesis of RNA has occurred; however, there is a marked increase in metabolic activity of the RNA as indicated by a substantial incorporation of 32P at this stage. The only significant departure in this correlation between the exchange of RKA phosphorus and amino acid incorporation is that the maximal turnover of Experimental
Cell Research 24
323
l&VA synthesis in the llyanassa embqo RSA phosphorus occurs masimal incorporation earlier. Iluring the first three visible organ primordia 10.000
during the fifth day of development, whereas the of leucine into the protein fraction occurs a day days of development of the flyrrr~rrsstr embryo no are formed as this period is chielly one of cleavage,
1
6000: 4000 1 30002000-
r\
loot-
\
- 1.o aI
"0 lOOO-
-0.9
2 s - 0.8 z : - 0.7 9 - 0.6 .; s -0.5 &
‘0, \
- 0.4 ,j -0.3 -0.2
‘0 -10 1 0
;
;
i
z
Days of development,
Fig. 2.--32P incorporation
into RNA
;
il
c 0 ;
. -0 0.1 g : r 0.0 7
18 C
and leucine-l-l%
incorporation
into protein.
determination and gastrulation. The marked changes in the metabolism of nucleic acids and proteins during the fourth day is not associated with gastrulation as this event is completed hy the end of the second day of development. The onset of RNA synthesis and the increase in incorporation of amino acids found during the fourth day precedes by one day the appearThe final two days of development consist ance of the organ primordia. of further elaboration and growth of the larval organs, and by the end of the seventh day a fully differentiated veliger larva is formed. As shown in Figs. 1 and 2, there is no significant synthesis of RX.4 nor incorporation of amino acids into the proteins during the last t\vo days of development.
J. X. Collier
324
DISCUSSION
The virtual absence of any incorporation of phosphorus into the RNA during the first two days reflects the passive role of this nucleic acid during the early development of the Ilyanassa embryo. Though the segregation of ribonucleoprotein to the various germinal regions during early cleavage may be of utmost importance for normal embryogenesis, the RNA at this stage appears to be inactive. This situation is analogous to that of the deoxyexcept at the time of their synthesis, and is what might be nucleoproteins, expected if the ribonucleoproteins serve as mediators of genetic information to the cytoplasm during development. The discrepancy observed between the time when the RN,4 of the Ilyanassa embryo begins to incorporate radioactive phosphorus and the period when the first synthesis of RNA occurs shows that the incorporation of 32P is an inadequate criterion for detecting the time of RNA synthesis. It is possible that the incorporation of phosphorus into the RNA observed during the third day is related to the apparent decrease in RNA found at that time. At present it is not possible to decide this point because of the uncertainty about the reality of this decrease in the RNA content. Otherwise the incorporation of 32P before any increase in RNA content may indicate that the RNA of the egg has begun to play some functional role in the embryo’s metabolism prior to the time when new RNA is synthesized. The close correlation between the incorporation of phosphorus in the RNA and the incorporation of 14C-leucine into the proteins strongly suggests that this is the case. As pointed out previously, there is relatively little synthetic activity during the early stages of development of the Ilyanassa embryo. In this embryo there is no increase in the incorporation of leucine into the proteins at the time of gastrulation. The small changes observed in the RNA content after the first day of development are of doubtful significance. There is no increase in the incorporation of 32P into the RNA during gastrulation, and this casts some doubt on the validity of the minor fluctuations in the RNA content reported above. As a tentative conclusion it appears that in the Ilyanasscr embryo there is very little synthetic activity at the time of embryonic determination, which occurs during early cleavage. The production of new RNA and proteins, which does not take place until the fourth day of development, begins intenselyjust before the onset of histological differentiation. Cohen [a] and Flickinger [5] found that at gastrulation there is a marked Experimental
Cell Research 24
KNL4 synthesis in the Ilyunassa embryo
325
increase in the incorporation of 14C-labeleci carbon from carbon dioxide into the proteins of the frog embryo. Hultin [9], using a cell-free system from the sea urchin embryo, studied the incorporation of ‘K-1 -1cucino into the proteins and found that incorporation began soon after fertilization anti culminated in the late blastula stage. Earlier, Hultin [lo ( had follo~vetl thtb incwqwration of 14C-lahelctl carbon dioxide into the proteins of the sea and had found that the specific activity of the carhoxyl urchin crnhyo reached a peak shortly before 111~ carbon incrcasetl just after fertilization, nnrwnc~hymc blastula stage, and increased again at the beginning of gastrul:ltion. \‘ille 11‘L 1 has reported on the uptake of 321’ into the RN>4 of the sea urchin embryo. From this data it appears that there is no increase in the incorporw tion 01’ I)hosphorus through the blastula stage, and any increase at the time of gastrulation is doubtful. Hultin [M-4] has studied the contributions 01 1”s labcllctl ammonium chloride, 14C-formate, 14(:-fornlatc, “(I-varbonatc and I*(:-acetate to Ihe RNA1 of the de\-eloping sea uwhin embryo, \vhcrcs tic found an intensified incorporation of labelled atonis from formale, carhonatc anti ammonium chloride during the blastula stage. The lahcllcct carbon from acetate \vas intensively incorporated during the mesrnch~mc~ I)lastula stage. Klson (It rrl. T-1 have sho\vn that an increase in the RX.-\ c~onl~~nl of the sea urchin embryo occur just before gastrulation, and ISSc-I
SUMMARY
The incorporation of 32P into the R?i,I and of nl.-leucinc-1 -14C into the proteins \vas determined at various developmental stages of the Ilytrnrt.sstr embryo. A significant incorporation of 321’ into the KS.4 owurrecl before any net synthesis of HSA.
J. H. Collier
326
A close correlation was found between the incorporation of phosphorus into the RI%A and the incorporation of 14C-leucine into the proteins. There was no increased protein or RNA metabolism associated with gastrulation. The period of intensive synthesis of protein and RNA was found to occur after embryonic determination and just before the beginning of histological differentiation. The author wishes to express his thanks to Marjorie M. Collier for her excellent assistance during the course of this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
B:~cKsTR~&~, S., A&iv 2001. 12, 339 (1959). COHEN, S., J. Biol. Chem., 211, 337 (1954). COLLIER, J. R., Exptl. Cell Research 21, 126 (1960). ELSON. D.. GUSTAFSON. T. and CHARGAFF, E., J. &I[. FLICKI’NGE~, R. A., Ex&l. Cell Research 6; 172 (1954). HULTIN, T., Arkiu Kemi 5, 267 (1953). __ Ibid. 6, 195 (1953).
Chem. 209, 285 (1954).
-__
Exptl. Cell Research 12, 518 (1957). Developmental Biol. 2, 61 (1960). HULTIN, T. grid WESSEL, G., Eiptl. Cell Research 3, 613 (1952). OGUR, hl. and ROSEN, G., Arch. Biochem. 25, 262 (1950).
VILLE,
C. A., LOWENS, M., GORDON, Physiol. 33, 93 (1949).
Experimenlal
Cell Research 24
M.,
LEOXARD,
E.
and
RICH,
A.,
.J. Cellular
Comp.