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Studies on the synthesis of ribonucleic acids in embryonic stages of Xenopus laevis
34
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95009
STUDIES ON THE SYNTHESIS OF RIBONUCLEIC ACIDS IN EMBRYONIC
STAGES OF XENOPUS
LAEVIS
M. D E C R O L Y , M. C A P E AS~) J. B R A C H E T
Labotatmm de Mt~vp~ologie ammale, Universitd libr: de Bru.~d:.~o~, 73vusseis (ilelgtum) (l
S U M M A I t tt
A study of the synthesis of ribonucleic acid in X'enopus taev/s ede.a ~.~ dffterent embryonic stages was andertaken. "['he base composition of the RNA synthesized after varying periods ot incubation in the presence of a~P-labeUed phosphate was ctetermined. RNA synthesis appears to bc.~in sooner tha~.l was ant,,cipated, namely j u , t a*ter fertilization, but it remains discrete during fht: early stages A c,ns:derabh, ,ncr~:a.~e occurred at gastrulation. The possibility that the newly synthesized RNA consists in ,mmle .-jr in Fart ot mes~en~or RNA is discussed
:' ,~R O D U C I IO .~
It ~.'ould t)e extrem,Sv intcrcsri;~g to discover tile molecular pr,;ce'-::,.'~iuvoJv.:¢: m cellulu c differentiation. .\ great deal c,f expe.rml..rmti work has s h o w n that , ,-..,,..,,u~!..:, ,.xi~t~ !)~:t~,een m o r p h o g e n e s i s
and
the ,;vnthesis of vncleic
~.clds
[t ',,- ~
::t~r ~: ,:.....
'. ,,xv;. that any modificatu,~, .: ":'ae R N , \ p, ~.iiy graci:"~t:~ (a:,znat-,,getati'. : .. v-~o-veutral) -- whether b y chemi~ :d or physical ,neaus (e.~; ,.~,~trif,:g:~k,.q) . !r, .', .:orious anomalies t (stro, g mic~,)cephaly, douoie en,Lryo:', ~:tc.; b, idles on the incorporation o~ 3~p in diflert-nt regions ~f tile ,'gi:;. cam,.,l ,,m i~ .~.-'.;UNDS AND FLICKINGER "~. haw; .:len:,mstrated ::q increas~ in the. cadioac~ ivity o~ ~h.~ nucleic acids in the induced ectoderm. The role of nucleic acids in pr,~tem synthesis has recently t)een greatly clanlied b y the work of Brenner and colleagues 8. They have :~ilown that m bacteria a special mtormatlonal RNA, called messenger RNA, c a m e s the genetic information possessect by DNA to the ribosomt~, tbc-mselves ttx~ sites of protein synthesis. Ie, the light ol these r~ults, BRACHET~,n has been able to propose several hypotheses which might explain what happrnq at a molecu!ar i~,vel during difIerentiarion, the synthesis ~.: r,;essem,~r R N A ' s would begin a: ;:a~:.truiat:an. "rl~is newly-turmed meusenger RNA :. ~,mld become attached to the ribasornes, ~t,,td~ preexist arm are already orgamze~ in eradients, thus transfornnn~ them into gradients ot polysoraes; as a result, protern avnthesis would be initiated .dong similar gradients, fhe Woc.'.uction of prO1;8ochim. Biophys...laa, 67 (I964) 3,f-39
SYNTHESIS OF RNA I~" Xenopus 2aevis
35
teins specific to each organ would follow from the competition set up between different ribosomes, charged with different messengers. Gastrulation is indeed thought to be an extremely important stage in embryonic development, for m a n y reasons. In 1951, STEINERT6 showed that, at least in the case of Rana/usca and Rana esculenta, net synthesis of RNA only becomes perceptible at gastrulation. According to DEUCHAR (personal communication) RNA synthesis would begin during cleavage in the species under consideration here: Xenopus laevis. But the degree of synthesis at this stage is, in any case, so low as to be negligible and one m a y even doubt its existence. Autoradiography enabled TENCER AND BIELIAVSKY7 to show that all the tritiated uridine and cvtidine taken up by embryos during cleavage, went into DNA and only entered RNA after gastrulation had begun. It is therefore clear that important metabolic changes occur at gastrulation. Studies on the effects of folic acid on morphogenesis also led GRANTs to the conclusion that new metabolic pathways become available for nucleic acids at gastrulation. It is also of significant interest that, in the case of lethal hybrids, embryonic development is blocked at late blastula or gastrula stages. All these experimental results corroborate one another and favour the hypothesis that the synthesis of messenger RNA might well begin at gastrulation. Moreover, GRANT'~* ~tudies on the incorporation of 32p and [14C]glycine into nucleic acids of Rana pipiens show that a high level of incorporation is obtained during the neurula stages. He also concluded that RNA has a rapid turnover as he observed a decline of incorporation into the RNA once the acid soluble fraction had equilibrated. We have undertaken a study of the synthesis of ribonucleic acid in Xenopus laevis eggs at different embryonic stages, and determined the base composition of RNA synthesized after varying periods of incubation in the presence of szP-labelled phosphate. The aim of these experiments was to attempt to detect the synthesis of a possible messenger ~RNA, and to determine the stage at which the synthesis might begin. MATERIAL AND METHODS
Xenopus laevis eggs obtained after injection with gonadotrope hormone (PregnylOrganon) were collected in tap water. The jelly was removed manually. Different stages were studied; 15 eggs for each stage were generally enough to measure the base composition of the newly-formed RNA. The eggs were exposed to a solution of 3~P-labelled phosphate, of known specific activity, for the desired period (2 h 3o rain; IO min; 3 rain). After incubation, the eggs were washed several times and homogenized at o °. Ttle nucleic acids and prot~4ns were then precipitated with o. 7 N perchloric acid containing H3PO 4. This was followed by a cold perchloric acid wash. The precipitate obtained was hydrolysed, in the presence of a carrier RNA, with o.3 N K O H at ",7° for 18 h. The alkaline hydrolysate was cooled to o ° and acidified with 2 N perchloric acid to precipitate the DNA and proteins. The clear supernatant was neutralized to p H 7 with K O H and the ribonucleotides were adsorbed on charcoal. The charcoal was then washed twice with potassium phosphate buffer (pH 7), and the adsorbed nucleotides were eluted x~dth alcohol-pyridine (5 : I, v/v). The eluate was evaporated to dryness in vacuum. The ribonucleotides were Biochim. Biophys. Acta, 87 (I964) 34-39
36
M. I)ECROI,Y, M. CAPE, J. BRACHET
dissolved in 2 ml of water and placed on a Dowex-I chloride column. The nucleotides were separated from each other by gradient elution with HC1 (o to o.I N) as eluent. The radioactivity of each fraction was determinated with a Geiger liquid counter (Adzam, type 18525). The ratio of the total radioactivity of the four nucleotidcs gave the relative composition of the newly-formed RNA on the eggs. RESULTS
The results obtained after a 2 . 5 - h pulse are given in Table I. They concern two different spawnings, A and B. For the sake of precision, the exact stages of development before exposure to 32p are indicated in the table of results, according to the specification of SHUMWAY~°. TABLE I RESULTS AFTI:.R A 2 . 5 - h
PULSE
Spawning A Stage
Co~egs RNA/egg
Spawning B
% C
% A
o"" G
% L'
Pull'y"
Cou~T.ts RNA/tgg
% C
% A
% G
% U
PulPy*
4 cells (Shumw a y 4)
3 II 4
2i.o
25. 9
30.4
22. 7
1.29
Morula (6)
7415
I7.O
35.6
25. 7
21. 7
1.32
3t4
22.i
27. 5
29.4
21.o
1.32
Blastula' (9)
12 121
2o.x
24. 7
33.6
25.6
1.27
529 499
22. 5 21.9
27. 3 26.5
28. 4 29. I
24.8 22. 5
1.26 1.25
Gastrula (12)
47 324
19.8
25.3
30.4
24.6
1.21
Medullary plate (14)
42 08. t
19.8
28.1
29.2
22.9
1.34
3032 4580 5429
26.1 23.3
27.(, 25.2
21.1
26. 5
25.9 29.4 28.9
20.3 22.I 23.6
1.I6 1.2o 1.23
Specific activity: x 8 o p C / m l 15 eggs into 2 ml
Specific activity: 5o /,C/ml 7 eggs into z ml
' P u ] P y expresses the ratio p u r i n e / p y r i m i d i n e .
Table I I groups all the results obtained experimentally on eggs from one single spawning, after a Io-min pulse. Only the number of counts per egg is given for the 3-min pulse, which was used for eggs from three different spawnings. The base composition obtained after such short pulses cannot be considered as significant, for reasons given later. The base composition of DNA in Xenopus laevis was also determined in order to compare it with that of the newly-synthesized R N A and to detect any eventual similarity between the two. The technique employed is that of FR~DgmC0 n in which only the concentration of adenylic acid is determined. The percentage of the other bases is calculated b y assuming that adenine = thymine; guanine =- cytosine. Biochim. Biophys. Acta, 87 (1964) 34-39
SYNTHESIS OF RNA 1N Xenopus laevis
37
TABLE II RESULTS AFTER A IO-MIN PULSE Counts RNA/egg
Stage
%C
% A
%G
% U
PulPy
8 cells (5)
238
23.x
24.7
28.7
23.5
I.I8
Morula (6)
155 386 249
23. 9 21.I 26.6
24.2 26.o 2o. 5
27.8 29.2 31.8
23.8 23. 4 21.2
x.I2 x.24 I.xo
Gastrula (io)(ix)
i593 20o 4
22.I 21. 5
25.9 27. 7
28.o 3I.O
23.8 20. 4
LI 7 1.39
Medullary plate (14)
2615
21.8
27. 7
27. 5
21.9
1.28
Base composition o[ DNA o] Xenopus %C
%A
%a
% T
Pu/ Py
24. 5
25. 5
24. 5
25. 5
x.oo
No significant difference was observed in the base composition of the newly formed RNA after a 2.5 h exposure to s2P-labelled phosphate. The slight differences recorded are well within the limits of experimental error. The most important fact which emerges from these experiments is the marked increase in s2p incorporation between the final gastrula and young neurula stages. The duration of the pulse is such that the nucleotide pool is likely to have reached equilibrium, and the base composition of the RNA may therefore be considered as validly representing the actual situation in the egg. There was quite a good deal of variation between different groups of eggs coming from different spawnings, but this marked increase in incorporation occurred in every case and coincided each time with the induction of the neural plate. When the duration of the szp treatment was cut down to ro rain, an increase in incorporation occurred during gastrulation. In Table I I I (3-min pulse) the number ot counts per minute is given, but not the base composition, since the duration of the pulse was too short to render the latter valid. Significant RN'A synthesis seems to begin when the medullary plate forms. TABLE III RESULTS AFTER A 3-MIN PULSE Counts RNA/egg
Stage Spawning No. •
Morula (6) Blastula (9) G a s t r u l a dorsal lip (xo) Mid-gastrula( xz )
Late gastrula (x2) Medullary plate (14) Neural t u b e
(x5)
Spawning No. a
7 47 x4 6
xo
312 478
133
Spawning No. 3
o o o
200 250
5oo * 963"
Specific activity: x m C / m l x5 eggs into 2 ml " 0.5 m C / m l x5 eggs into 2 ml
Biochim. Biophys. Acta, 87 (1964) 34-39
38
M. DECROLY, M. CAPE, J. BRACHET
The fact that the differences in incorporation into RNA are not due to differences in the degree of uptake of szp should also be stressed. Measurement of the acid-soluble fraction revealed considerable similitude, whatever stage was studied.
DISCUSSION The most important conclusionwhich can be drawn from this work is the net increase in labelling which coincides with neurulation, whatever the dmation of the pulse. Nevertheless, s,p incorporation begins during cleavage. Moreover it seems that, if introduced into the amphibim~ egg by microinj,?ction, ['4C]leucine can bo incorporated into the egg proteins from the two-cell stage of:wards'z. Unfortunately it has not been possible to ea]<'a:.~tethe rate and level of synthesis of the newly-synthesizedRNA since the experiments described h~re required the use of carrier ~ N A . Is tb,: ,:ewly synthesized RNA messenger R N A : Befort. answering thl~ q,testion, we have t,, ,ind out whether it is of nuclear origin. Although total RNA was extracted, thmt~ mc mmly reason: ",or bchevmg that the newly-synthesized molecules :,r,' ;)de(,d ,.)f nuclcc: origin. Fi,stly, T2NCI,:R ~XI.~ BIEL1AVSKY~ have shown that up t,. ,~.ulula ~:av, , uridine and cytidine are only incorporate.~ into the nucleus. Otht-: aatLor.¢. :ising CO, as precursor, have also rec,'rded the predominance of mlt:lc:~, ,\'el cytoplasmic labelling np to the stage of neurulala, 14. Moreover, it has been welt est,ablished that the synthesis of RNA, using. DNA as a template, is blocked by actinomycin D 's. Now, BR.~CHET AND 1)ENXS~6 observed a strong inhibitory effect of this substance on embty:mic development, particularly on dorso-ventral and eephalo-caudal differentiation. The fact that this specific inhibition takes effect during gastrulation nece~arily implies that N N A is synthesized in the nucleu~ during neural induc;:ion. The blocking of nervous differentiation under the influence of actinomycin D has been studied b y DEmS ~', usin~ :,n ,~xplant .~ystem in which the orgamzmg antt the inducing tissue arc isolated from the rest of i he embryo. Both the competence of the ectoblast and the inducing power, ,l the organizer were destroyed by actinomycm D. Autoradmgraphy enabled D E m s to demonstrate tl:e almost complete suppression of RNA synthesis in the nucleus or ecto.termal .'ells irrupted x:ti', actmon.vcin D. Pulse experimer, ts using ~"P in tbe presence ot aetinomycin D are being carriec out in our laboratory. But the experimental results assembled to (late all point to the conclusion that the RN.'k which is labelled is located in the nucleus. The second important aspect to consider is the nature of the base composition of the newly-synthesized RIgA as compared wath DNA. Ext)erimcnt:dly. no simi/urity has been observed. But il now seems plausible that messenger RNA need not necessarily have the same bas,: composition as DNA; this is no longer thought to be a conaitio sine qua non for the recognition of informational RNA, since in organisms showing a high level of differentiation it la probable that only part of the genome is functional xs. The newly synthesized RNA is most probably composed of several ddferent kinds of R N A ' s having similar rates *)f turnover. Table i reveals the close similarity between the base composition obsera, ed and that recorded for ribosomes. Synthesis of ribosomal R N A m a y occur simultaneously. Extensive studies on the synthesis of ;3tochim. B,ophys. A.cta 8- ¢1064) 34-39
SYNTHESIS OF R N A IN Xenopus laevis
39
ribosomes in R a n a pipiens, b y BROWN AND CASTONTM, h a v e led these a u t h o r s to t h e conclusion t h a t y o u n g e m b r y o s h a v e a v e r y low ribosome c o n t e n t , a n d t h a t ribosome synthesis does n o t begin before t h e stage at w h i c h m u s c u l a r c o n t r a c t i o n first occurs. H o w e v e r , p r e l i m i n a r y e x p e r i m e n t s carried o u t in this l a b o r a t o r y show a " r i b o somal f r a c t i o n " rich in R N A ; in addition this fraction has the highest R N A / p r o t e i n ratio, even in the y o u n g gastrula. In conclusion, R N A synthesis appears to begin sooner t h a n was a n t i c i p a t e d , t h a t is to say, iust a f t e r fertilization, a l t h o u g h it remains discrete d u r i n g the early stages. A considerable increase in the rate of synthesis occurs at gastrulation. A t neurulation, it is still more intense. T h e base composition of the RNA. synthesized remains the same t h r o u g h o u t d e v e l o p m e n t , a n d bears a g r eat er resemblancc to that ge n er al l y found in ribosomes than in D N A . ACKNOWLEDGEMENTS
This investigation was supported by Euratom (Contract o16-61-1o ABIB). We wish to thank Mine P. MALPOIX for the translation of the text. REFERENCES
' J. F4aACIIET, The Biochemistry o/ Development, Pergamon Press, London, 196o, p. 174. E. I¢.OUNDSAND A. FLICKXr~GER,.[. Exptl. Zool., z37 (x958) 479. 3 S. BRENNER, F. JACOB AND I~I. ,'~.[~-SELSON, Nature, i9o (1961) 576. 4 j. BRACHET,J. Celt. Comp. Physiol., suppl, x, 60 (1962) i. 6 j. BRACHET, N. BIEUAVSKYAND R. TENCER, Bull. Acad. Roy. "]'[ed. Belg., 48 (I962) 255. 6 M. STEINERT, Biochim. Biophys. Acta, x8 (1955) 51x. R. TENCER AND N. BIELIAVSKV,Exptl. Cell. Res., 2x (I96O) 279. P. Gr~ANr, Develop. Biol., 2 (x96o) x97. 0 p. GRANT, J. Cell. Comp. Physiol., 52 (1958) 249. ,0 W. SHUMWAV,Anat. Record, 78 (,94 o) I39. n E. FR~DgRICg, H. OTa AND F. FONTAXNE,J. Mol. Biol., 3 (i96i) I1. ,2 E. VAN STEVENS, personal communication. la A. FIcQ, Experienta, lO (1954) zo. l, R. TENCER, J. Embryol. Exptl. Morph., 6 (I958) ~I 7. i6 E. REICti, [. H. GOLI)BERG AND .~1. I~ABINOWITZ, :Valt~re, I 9 6 ( i 9 6 2 ) 743. t6 j. BRACHET ^ND H. DEmS, Nature, I08 (1963) 205 . ,7 H. DEv'~s, Exptl. Cell Res., 3° (1963) 613. is M. ERRERA, Confrrence faite 5. la ,l.ze rrunion de la Soc. de Biochimie, I962. ,t D. D. BROWNANn J. D. CASTO,n, Devel,:p. Biol., 5 (I962) 412. Biochim. Biophys. Acta, 87 (x964) 34-39
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95009
STUDIES ON THE SYNTHESIS OF RIBONUCLEIC ACIDS IN EMBRYONIC
STAGES OF XENOPUS
LAEVIS
M. D E C R O L Y , M. C A P E AS~) J. B R A C H E T
Labotatmm de Mt~vp~ologie ammale, Universitd libr: de Bru.~d:.~o~, 73vusseis (ilelgtum) (l
S U M M A I t tt
A study of the synthesis of ribonucleic acid in X'enopus taev/s ede.a ~.~ dffterent embryonic stages was andertaken. "['he base composition of the RNA synthesized after varying periods ot incubation in the presence of a~P-labeUed phosphate was ctetermined. RNA synthesis appears to bc.~in sooner tha~.l was ant,,cipated, namely j u , t a*ter fertilization, but it remains discrete during fht: early stages A c,ns:derabh, ,ncr~:a.~e occurred at gastrulation. The possibility that the newly synthesized RNA consists in ,mmle .-jr in Fart ot mes~en~or RNA is discussed
:' ,~R O D U C I IO .~
It ~.'ould t)e extrem,Sv intcrcsri;~g to discover tile molecular pr,;ce'-::,.'~iuvoJv.:¢: m cellulu c differentiation. .\ great deal c,f expe.rml..rmti work has s h o w n that , ,-..,,..,,u~!..:, ,.xi~t~ !)~:t~,een m o r p h o g e n e s i s
and
the ,;vnthesis of vncleic
~.clds
[t ',,- ~
::t~r ~: ,:.....
'. ,,xv;. that any modificatu,~, .: ":'ae R N , \ p, ~.iiy graci:"~t:~ (a:,znat-,,getati'. : .. v-~o-veutral) -- whether b y chemi~ :d or physical ,neaus (e.~; ,.~,~trif,:g:~k,.q) . !r, .', .:orious anomalies t (stro, g mic~,)cephaly, douoie en,Lryo:', ~:tc.; b, idles on the incorporation o~ 3~p in diflert-nt regions ~f tile ,'gi:;. cam,.,l ,,m i~ .~.-'.;UNDS AND FLICKINGER "~. haw; .:len:,mstrated ::q increas~ in the. cadioac~ ivity o~ ~h.~ nucleic acids in the induced ectoderm. The role of nucleic acids in pr,~tem synthesis has recently t)een greatly clanlied b y the work of Brenner and colleagues 8. They have :~ilown that m bacteria a special mtormatlonal RNA, called messenger RNA, c a m e s the genetic information possessect by DNA to the ribosomt~, tbc-mselves ttx~ sites of protein synthesis. Ie, the light ol these r~ults, BRACHET~,n has been able to propose several hypotheses which might explain what happrnq at a molecu!ar i~,vel during difIerentiarion, the synthesis ~.: r,;essem,~r R N A ' s would begin a: ;:a~:.truiat:an. "rl~is newly-turmed meusenger RNA :. ~,mld become attached to the ribasornes, ~t,,td~ preexist arm are already orgamze~ in eradients, thus transfornnn~ them into gradients ot polysoraes; as a result, protern avnthesis would be initiated .dong similar gradients, fhe Woc.'.uction of prO1;8ochim. Biophys...laa, 67 (I964) 3,f-39
SYNTHESIS OF RNA I~" Xenopus 2aevis
35
teins specific to each organ would follow from the competition set up between different ribosomes, charged with different messengers. Gastrulation is indeed thought to be an extremely important stage in embryonic development, for m a n y reasons. In 1951, STEINERT6 showed that, at least in the case of Rana/usca and Rana esculenta, net synthesis of RNA only becomes perceptible at gastrulation. According to DEUCHAR (personal communication) RNA synthesis would begin during cleavage in the species under consideration here: Xenopus laevis. But the degree of synthesis at this stage is, in any case, so low as to be negligible and one m a y even doubt its existence. Autoradiography enabled TENCER AND BIELIAVSKY7 to show that all the tritiated uridine and cvtidine taken up by embryos during cleavage, went into DNA and only entered RNA after gastrulation had begun. It is therefore clear that important metabolic changes occur at gastrulation. Studies on the effects of folic acid on morphogenesis also led GRANTs to the conclusion that new metabolic pathways become available for nucleic acids at gastrulation. It is also of significant interest that, in the case of lethal hybrids, embryonic development is blocked at late blastula or gastrula stages. All these experimental results corroborate one another and favour the hypothesis that the synthesis of messenger RNA might well begin at gastrulation. Moreover, GRANT'~* ~tudies on the incorporation of 32p and [14C]glycine into nucleic acids of Rana pipiens show that a high level of incorporation is obtained during the neurula stages. He also concluded that RNA has a rapid turnover as he observed a decline of incorporation into the RNA once the acid soluble fraction had equilibrated. We have undertaken a study of the synthesis of ribonucleic acid in Xenopus laevis eggs at different embryonic stages, and determined the base composition of RNA synthesized after varying periods of incubation in the presence of szP-labelled phosphate. The aim of these experiments was to attempt to detect the synthesis of a possible messenger ~RNA, and to determine the stage at which the synthesis might begin. MATERIAL AND METHODS
Xenopus laevis eggs obtained after injection with gonadotrope hormone (PregnylOrganon) were collected in tap water. The jelly was removed manually. Different stages were studied; 15 eggs for each stage were generally enough to measure the base composition of the newly-formed RNA. The eggs were exposed to a solution of 3~P-labelled phosphate, of known specific activity, for the desired period (2 h 3o rain; IO min; 3 rain). After incubation, the eggs were washed several times and homogenized at o °. Ttle nucleic acids and prot~4ns were then precipitated with o. 7 N perchloric acid containing H3PO 4. This was followed by a cold perchloric acid wash. The precipitate obtained was hydrolysed, in the presence of a carrier RNA, with o.3 N K O H at ",7° for 18 h. The alkaline hydrolysate was cooled to o ° and acidified with 2 N perchloric acid to precipitate the DNA and proteins. The clear supernatant was neutralized to p H 7 with K O H and the ribonucleotides were adsorbed on charcoal. The charcoal was then washed twice with potassium phosphate buffer (pH 7), and the adsorbed nucleotides were eluted x~dth alcohol-pyridine (5 : I, v/v). The eluate was evaporated to dryness in vacuum. The ribonucleotides were Biochim. Biophys. Acta, 87 (I964) 34-39
36
M. I)ECROI,Y, M. CAPE, J. BRACHET
dissolved in 2 ml of water and placed on a Dowex-I chloride column. The nucleotides were separated from each other by gradient elution with HC1 (o to o.I N) as eluent. The radioactivity of each fraction was determinated with a Geiger liquid counter (Adzam, type 18525). The ratio of the total radioactivity of the four nucleotidcs gave the relative composition of the newly-formed RNA on the eggs. RESULTS
The results obtained after a 2 . 5 - h pulse are given in Table I. They concern two different spawnings, A and B. For the sake of precision, the exact stages of development before exposure to 32p are indicated in the table of results, according to the specification of SHUMWAY~°. TABLE I RESULTS AFTI:.R A 2 . 5 - h
PULSE
Spawning A Stage
Co~egs RNA/egg
Spawning B
% C
% A
o"" G
% L'
Pull'y"
Cou~T.ts RNA/tgg
% C
% A
% G
% U
PulPy*
4 cells (Shumw a y 4)
3 II 4
2i.o
25. 9
30.4
22. 7
1.29
Morula (6)
7415
I7.O
35.6
25. 7
21. 7
1.32
3t4
22.i
27. 5
29.4
21.o
1.32
Blastula' (9)
12 121
2o.x
24. 7
33.6
25.6
1.27
529 499
22. 5 21.9
27. 3 26.5
28. 4 29. I
24.8 22. 5
1.26 1.25
Gastrula (12)
47 324
19.8
25.3
30.4
24.6
1.21
Medullary plate (14)
42 08. t
19.8
28.1
29.2
22.9
1.34
3032 4580 5429
26.1 23.3
27.(, 25.2
21.1
26. 5
25.9 29.4 28.9
20.3 22.I 23.6
1.I6 1.2o 1.23
Specific activity: x 8 o p C / m l 15 eggs into 2 ml
Specific activity: 5o /,C/ml 7 eggs into z ml
' P u ] P y expresses the ratio p u r i n e / p y r i m i d i n e .
Table I I groups all the results obtained experimentally on eggs from one single spawning, after a Io-min pulse. Only the number of counts per egg is given for the 3-min pulse, which was used for eggs from three different spawnings. The base composition obtained after such short pulses cannot be considered as significant, for reasons given later. The base composition of DNA in Xenopus laevis was also determined in order to compare it with that of the newly-synthesized R N A and to detect any eventual similarity between the two. The technique employed is that of FR~DgmC0 n in which only the concentration of adenylic acid is determined. The percentage of the other bases is calculated b y assuming that adenine = thymine; guanine =- cytosine. Biochim. Biophys. Acta, 87 (1964) 34-39
SYNTHESIS OF RNA 1N Xenopus laevis
37
TABLE II RESULTS AFTER A IO-MIN PULSE Counts RNA/egg
Stage
%C
% A
%G
% U
PulPy
8 cells (5)
238
23.x
24.7
28.7
23.5
I.I8
Morula (6)
155 386 249
23. 9 21.I 26.6
24.2 26.o 2o. 5
27.8 29.2 31.8
23.8 23. 4 21.2
x.I2 x.24 I.xo
Gastrula (io)(ix)
i593 20o 4
22.I 21. 5
25.9 27. 7
28.o 3I.O
23.8 20. 4
LI 7 1.39
Medullary plate (14)
2615
21.8
27. 7
27. 5
21.9
1.28
Base composition o[ DNA o] Xenopus %C
%A
%a
% T
Pu/ Py
24. 5
25. 5
24. 5
25. 5
x.oo
No significant difference was observed in the base composition of the newly formed RNA after a 2.5 h exposure to s2P-labelled phosphate. The slight differences recorded are well within the limits of experimental error. The most important fact which emerges from these experiments is the marked increase in s2p incorporation between the final gastrula and young neurula stages. The duration of the pulse is such that the nucleotide pool is likely to have reached equilibrium, and the base composition of the RNA may therefore be considered as validly representing the actual situation in the egg. There was quite a good deal of variation between different groups of eggs coming from different spawnings, but this marked increase in incorporation occurred in every case and coincided each time with the induction of the neural plate. When the duration of the szp treatment was cut down to ro rain, an increase in incorporation occurred during gastrulation. In Table I I I (3-min pulse) the number ot counts per minute is given, but not the base composition, since the duration of the pulse was too short to render the latter valid. Significant RN'A synthesis seems to begin when the medullary plate forms. TABLE III RESULTS AFTER A 3-MIN PULSE Counts RNA/egg
Stage Spawning No. •
Morula (6) Blastula (9) G a s t r u l a dorsal lip (xo) Mid-gastrula( xz )
Late gastrula (x2) Medullary plate (14) Neural t u b e
(x5)
Spawning No. a
7 47 x4 6
xo
312 478
133
Spawning No. 3
o o o
200 250
5oo * 963"
Specific activity: x m C / m l x5 eggs into 2 ml " 0.5 m C / m l x5 eggs into 2 ml
Biochim. Biophys. Acta, 87 (1964) 34-39
38
M. DECROLY, M. CAPE, J. BRACHET
The fact that the differences in incorporation into RNA are not due to differences in the degree of uptake of szp should also be stressed. Measurement of the acid-soluble fraction revealed considerable similitude, whatever stage was studied.
DISCUSSION The most important conclusionwhich can be drawn from this work is the net increase in labelling which coincides with neurulation, whatever the dmation of the pulse. Nevertheless, s,p incorporation begins during cleavage. Moreover it seems that, if introduced into the amphibim~ egg by microinj,?ction, ['4C]leucine can bo incorporated into the egg proteins from the two-cell stage of:wards'z. Unfortunately it has not been possible to ea]<'a:.~tethe rate and level of synthesis of the newly-synthesizedRNA since the experiments described h~re required the use of carrier ~ N A . Is tb,: ,:ewly synthesized RNA messenger R N A : Befort. answering thl~ q,testion, we have t,, ,ind out whether it is of nuclear origin. Although total RNA was extracted, thmt~ mc mmly reason: ",or bchevmg that the newly-synthesized molecules :,r,' ;)de(,d ,.)f nuclcc: origin. Fi,stly, T2NCI,:R ~XI.~ BIEL1AVSKY~ have shown that up t,. ,~.ulula ~:av, , uridine and cytidine are only incorporate.~ into the nucleus. Otht-: aatLor.¢. :ising CO, as precursor, have also rec,'rded the predominance of mlt:lc:~, ,\'el cytoplasmic labelling np to the stage of neurulala, 14. Moreover, it has been welt est,ablished that the synthesis of RNA, using. DNA as a template, is blocked by actinomycin D 's. Now, BR.~CHET AND 1)ENXS~6 observed a strong inhibitory effect of this substance on embty:mic development, particularly on dorso-ventral and eephalo-caudal differentiation. The fact that this specific inhibition takes effect during gastrulation nece~arily implies that N N A is synthesized in the nucleu~ during neural induc;:ion. The blocking of nervous differentiation under the influence of actinomycin D has been studied b y DEmS ~', usin~ :,n ,~xplant .~ystem in which the orgamzmg antt the inducing tissue arc isolated from the rest of i he embryo. Both the competence of the ectoblast and the inducing power, ,l the organizer were destroyed by actinomycm D. Autoradmgraphy enabled D E m s to demonstrate tl:e almost complete suppression of RNA synthesis in the nucleus or ecto.termal .'ells irrupted x:ti', actmon.vcin D. Pulse experimer, ts using ~"P in tbe presence ot aetinomycin D are being carriec out in our laboratory. But the experimental results assembled to (late all point to the conclusion that the RN.'k which is labelled is located in the nucleus. The second important aspect to consider is the nature of the base composition of the newly-synthesized RIgA as compared wath DNA. Ext)erimcnt:dly. no simi/urity has been observed. But il now seems plausible that messenger RNA need not necessarily have the same bas,: composition as DNA; this is no longer thought to be a conaitio sine qua non for the recognition of informational RNA, since in organisms showing a high level of differentiation it la probable that only part of the genome is functional xs. The newly synthesized RNA is most probably composed of several ddferent kinds of R N A ' s having similar rates *)f turnover. Table i reveals the close similarity between the base composition obsera, ed and that recorded for ribosomes. Synthesis of ribosomal R N A m a y occur simultaneously. Extensive studies on the synthesis of ;3tochim. B,ophys. A.cta 8- ¢1064) 34-39
SYNTHESIS OF R N A IN Xenopus laevis
39
ribosomes in R a n a pipiens, b y BROWN AND CASTONTM, h a v e led these a u t h o r s to t h e conclusion t h a t y o u n g e m b r y o s h a v e a v e r y low ribosome c o n t e n t , a n d t h a t ribosome synthesis does n o t begin before t h e stage at w h i c h m u s c u l a r c o n t r a c t i o n first occurs. H o w e v e r , p r e l i m i n a r y e x p e r i m e n t s carried o u t in this l a b o r a t o r y show a " r i b o somal f r a c t i o n " rich in R N A ; in addition this fraction has the highest R N A / p r o t e i n ratio, even in the y o u n g gastrula. In conclusion, R N A synthesis appears to begin sooner t h a n was a n t i c i p a t e d , t h a t is to say, iust a f t e r fertilization, a l t h o u g h it remains discrete d u r i n g the early stages. A considerable increase in the rate of synthesis occurs at gastrulation. A t neurulation, it is still more intense. T h e base composition of the RNA. synthesized remains the same t h r o u g h o u t d e v e l o p m e n t , a n d bears a g r eat er resemblancc to that ge n er al l y found in ribosomes than in D N A . ACKNOWLEDGEMENTS
This investigation was supported by Euratom (Contract o16-61-1o ABIB). We wish to thank Mine P. MALPOIX for the translation of the text. REFERENCES
' J. F4aACIIET, The Biochemistry o/ Development, Pergamon Press, London, 196o, p. 174. E. I¢.OUNDSAND A. FLICKXr~GER,.[. Exptl. Zool., z37 (x958) 479. 3 S. BRENNER, F. JACOB AND I~I. ,'~.[~-SELSON, Nature, i9o (1961) 576. 4 j. BRACHET,J. Celt. Comp. Physiol., suppl, x, 60 (1962) i. 6 j. BRACHET, N. BIEUAVSKYAND R. TENCER, Bull. Acad. Roy. "]'[ed. Belg., 48 (I962) 255. 6 M. STEINERT, Biochim. Biophys. Acta, x8 (1955) 51x. R. TENCER AND N. BIELIAVSKV,Exptl. Cell. Res., 2x (I96O) 279. P. Gr~ANr, Develop. Biol., 2 (x96o) x97. 0 p. GRANT, J. Cell. Comp. Physiol., 52 (1958) 249. ,0 W. SHUMWAV,Anat. Record, 78 (,94 o) I39. n E. FR~DgRICg, H. OTa AND F. FONTAXNE,J. Mol. Biol., 3 (i96i) I1. ,2 E. VAN STEVENS, personal communication. la A. FIcQ, Experienta, lO (1954) zo. l, R. TENCER, J. Embryol. Exptl. Morph., 6 (I958) ~I 7. i6 E. REICti, [. H. GOLI)BERG AND .~1. I~ABINOWITZ, :Valt~re, I 9 6 ( i 9 6 2 ) 743. t6 j. BRACHET ^ND H. DEmS, Nature, I08 (1963) 205 . ,7 H. DEv'~s, Exptl. Cell Res., 3° (1963) 613. is M. ERRERA, Confrrence faite 5. la ,l.ze rrunion de la Soc. de Biochimie, I962. ,t D. D. BROWNANn J. D. CASTO,n, Devel,:p. Biol., 5 (I962) 412. Biochim. Biophys. Acta, 87 (x964) 34-39