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Expression of ribosomal-protein genes in Xenopus laevis development
Cell, Vol. 30. 163-I
71, August
1982,
Copyright
0 1982
by MIT
Expression of Ribosomal-Protein Genes in Xenopus laevis Development Paola Pierandrei-Amaldi and Nadia lstituto di Biologia Cellulare, C.N.R. Via Romagnosi 18/A 00196 Rome, Italy Elena Beccari, Irene Bozzoni and Francesco Amaldi Centro Acidi Nucleici, C.N.R. lstituto di Fisiologia Generale Universita di Roma 00185 Rome Italy
Campioni
Summary Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 24, become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs. Introduction The process of ribosome biosynthesis includes a number of reactions that together lead to the simultaneous and balanced production of the ribosomal components: the two large rRNAs, the 5s RNA and the ribosomal proteins. The mechanisms involved in the regulated expression of the ribosomal proteins and of the rRNA are fairly well understood in procaryotic systems (Nomura and Post, 1980) but are still not clear in eucaryotes. So far the regulation of ribosomalprotein (r-protein) and rRNA synthesis has been investigated in yeast (Gorenstein and Warner, 1976; Rosbash et al., 1981), in cultured animal cells (Craig and Perry, 1971; Warner, 1977; Geyer et al., 1982) and in regenerating rat liver (Faliks and Meyuhas,
1982). Although much has been learned, different results have been obtained in various systems and under different conditions, so that a general picture cannot be drawn (for review see Warner et al., 1980). We are interested in the Xenopus laevis system because it presents two physiological situations in which a change in the production of ribosomes occurs: oogenesis and early embryogenesis. In both cases a large amount of ribosomes is accumulated; the genes that encode the ribosomal proteins are therefore expected to be particularly active. An X. laevis oocyte cDNA bank was constructed with an mRNA fraction enriched for r-protein-coding capacity (Pierandrei-Amaldi and Beccari, 1980; Bozzoni et al., 1981). Clones carrying sequences for six different ribosomal proteins (Sl , S8, S19, Ll , L14 and L32) were selected by positive translation of mRNA specifically hybridized to the recombinant plasmids. We have used the recombinant clones as probes to monitor the behavior of specific r-protein mRNAs during oogenesis and embryogenesis. The pattern of accumulation of these mRNAs was followed and was related to the pattern of r-protein synthesis during embryogenesis. Anucleolate mutants were used to study the influence of rRNA on the expression of rprotein genes both at the level of mRNA production and at the level of r-protein synthesis. Results The sizes of the mRNA sequences specific for six ribosomal proteins were analyzed in X. laevis oocytes that had not yet completed vitellogenesis. Poly(A)+ RNA from these oocytes (Dumont stages I-III) has a good coding capacity for ribosomal proteins when tested in vitro. An enrichment for r-protein mRNA was obtained by selection of a 10-16s sucrose gradient fraction of the poly(A)+ RNA. It has been estimated that, all together, the 70 ribosomal proteins account for IO%-20% of the products coded in vitro by this mRNA fraction (Pierandrei-Amaldi and Beccari, 1980). Each mRNA sequence is thus present at a level of about 0.2%. Enriched poly(A)+ RNA was run in agarose gels, transferred to DBM paper or to nitrocellulose filters and hybridized to the six cloned cDNA probes for the different ribosomal proteins. The calibration was made with denatured RNAs as molecular weight standards: 28S, 18S, 5s and 4s RNAs from X. laevis and 9s RNA from rabbit reticulocyte; labeled denatured +X174 Hae Ill digestion fragments were also used as a reference. The average values of different experiments are reported in Table 1. The mRNA size is proportional to the protein length as determined on SDS-acrylamide gels (PierandreiAmaldi and Beccari, 1980). Accumulation of r-Protein mRNA during Oogenesis It was of interest to follow the amount of mRNA specific for ribosomal proteins during the course of oo-
Table 1. Length of Some r-Protein Previtellogenic X-laevis Oocytes
mRNA Sequences
in
Ribosomal Protein
Molecular Weight (Dalton&
Expected Length of mRNACoding Region (Nucleotides)
Ll
54,000
1,400
1,500
L14
20,800
540
750
pXom92
L32
8,800
230
550
pXom78
Sl
32,000
850
1,000
pXom91
S8
21,500
560
850
pXom62
s19
12.500
320
650
pXom69
Measured Length of mRNA (Nucleotides)
Recombinant Plasmid pXomlO2
Ribosomal proteins were numbered and tiieir molecular weight was calculated as described by Pierandrei-Amaldi and Beccari (1980). From the sequence of r-protein cDNAs (Amaldi et al., 19821, an average length of 115 amino acids was calculated. RNA length was determined on agarose gels; poly(A)+ RNA was transferred to DBM paper or nitrocellulose filters and probed with labeled cloned cDNA sequences.
genesis, in connection with the known features of rRNA synthesis. After collagenase digestion of the ovary walls, the free oocytes were washed several times to eliminate debris and were staged under a microscope into completely transparent oocytes, less than 250 pm in diameter (Dumont early I) (stage A); fully transparent, well visible nucleus, 250-300 pm (Dumont late I) (stage B); light yellow, round, nucleus not visible, 300-350 pm (Dumont II) (stage C); yellowbrown, starting pigmentation, 400-500 pm (Dumont Ill) (stage D); brown, half-size, about 600 pm (Dumont IV) (stage E); fully mature, 1.2-1.3 mm (Dumont VI) (stage F). Total RNA was extracted from 25 oocytes of each group, and amounts corresponding to ten oocytes were run on agarose gels under denaturing conditions and hybridized to the r-protein probes. The autoradiographs in Figure 1 show a reproducible increase of signal intensity from stages A to B to C and a stable level through stages D and E (Figures 1 a and 1 b). In fully matured oocytes the hybridizing signal was reduced. To rule out artifacts due to RNA overloading, we repeated the experiment with RNA corresponding to only two oocytes per lane, with identical results (Figure 1 c). Accumulation of r-Protein mRNA in the Embryo The accumulation of individual r-protein mRNA has been followed in early embryogenesis. To see the onset of r-protein mRNA synthesis, we analyzed several developmental stages from two blastomeres, up to the tailbud stage (stages 30-33). Cytoplasmic RNA was extracted from a fixed number of staged embryos (Nieuwkoop and Faber, 1956) generally 100, and the RNA corresponding to ten embryos was run on agarose gels under denaturing conditions. The amount of
ABCDEF Figure
1. Accumulation
of r-protein
mRNA during
Oogenesis.
Total RNA corresponding to ten (a and b) or two (c) oocytes of each stage was denatured, run on agarose gels and blotted on nitrocellulose filters. Filters were hybridized to “P-labeled pXom102 (ribosomal protein Ll) (a) or to 32P-labeled pXom92 (ribosomal protein L14) (b and c). (Lanes A-F) RNA isolated from corresponding oocyte stages (see text).
cytoplasmic RNA per embryo, mostly rRNA of maternal origin, was fairly constant in the period of time examined, about 3.5 pg. Only in the last two stages was a slight but reproducible increase observed. RNA was transferred from agarose to nitrocellulose membranes for hybridization with 3”P-nick-translated r-protein-specific probes. Figure 2 shows the pattern of accumulation of mRNA for ribosomal proteins L14 (Figure 2a) and Si (Figure 2b) as revealed by hybridization with the corresponding probes. mRNA starts to be detectable clearly at stages 10-l 1 (gastrula) and increases later on. A quantitative evaluation of the amount of mRNA present at different stages has been made by densitometric scanning of the autoradiographs. We have calculated the relative amount of mRNA per cell, taking into consideration the increasing number of cells per embryo (Figure 3). The amount of L14 and Sl mRNA increased rapidly between stages 10 and 16, and remained constant afterwards. A similar pattern was also observed for mRNAs of proteins Ll and S19 when hybridized to the corresponding probes (data not shown). In Figures 2a and 2b a faint hybridization band of slightly lower molecular weight can be seen at stages 2 and 5. This band almost disappears in the subsequent cleavage stages. The mRNA present at the beginning of embryogenesis probably represents residual maternal mRNA, and the slight difference in molecular weight may be due to shorter poly(A) tracts (Sagata et al., 1980). The probe for L14 mRNA was also hybridized to poly(A)+ RNA from embryos of different stages. Results similar to those for total RNA were obtained, including the slowmigrating band at early cleavage.
Ribosomal-Protein 165
Synthesis
in Xenopus
Development
a
oocyte
2
5
9
7
10
11
12
16
21
24
1
Figure
2. Accumulation
of r-Protein
mRNA
33 i
during
Embryogenesis
Cytoplasmic RNA derived from ten embryos of each stage was denatured, run on agarose gels and transferred to nitrocellulose filters as described in the Experimental Procedures. Filters were hybridized to ‘*P-labeled pXom92 (ribosomal protein L14) (a) or to pXom91 (ribosomal protein Si) (b). Numbers of lanes: embryo stages (Nieuwkoop and Faber, 1956). (Lane oocyte) cytoplasmic RNA from oocyte stages IV-VI (Dumont, 1972). In (b), stage 2 is missing.
Figure 3. Relative ogenesis
Amount
of r-Protein
mRNA
per Cell during
Embry-
The extent of hybridization of the r-protein (r-p) mRNA to the corresponding probes was estimated by densitometric tracing of the autoradiographs shown in Figure 2. (O- - -0) mRNA for ribosomal protein L14; (0- - -0) mRNA for ribosomal protein Sl The values have been normalized to the number of cells per embryo of the specific stage (Gurdon, 1974). Curves have been extrapolated to the last point. (A-A) Extraction yields of RNA from the different developmental stages.
Synthesis of Ribosomal Proteins in the Embryo To correlate the accumulation pattern of r-protein mRNAs with their translation, we followed the incorporation of 35S-methionine into ribosomal proteins during early embryogenesis. Embryos from early
cleavage to tadpole stages (stage 2-5, 7-9, 1 O-l 2, 14-16, 22-24, 26-27, 29-31 and 41) were labeled with 35S-methionine for 5-6 hr at 22°C. To circumvent the permeability barrier of the embryos, we tried different methods: bisection of embryos (Bachvarova and Davidson, 1966), EDTA treatment before incubation (Landesman and Gross, 1968) and injection with glass microneedles. The three methods gave the same qualitative results, but injection was chosen because the efficiency of incorporation was good and the embryos were not seriously damaged. Homogenates of incubated embryos were extracted with acetic acid and precipitated with acetone. Ribosomal proteins were then separated from total ceil proteins by two-dimensional electrophoresis (Gorenstein and Warner, 1976). Gels were loaded with extracts corresponding to four labeled embryos. Figure 4 shows examples of the fluorographs of the two-dimensional patterns of ribosomal proteins synthesized by embryos of different stages. Since, as mentioned, each gel was loaded with material from an equal number of embryos labeled under standard conditions, comparison of the patterns is possible. Moreover, histones (see below) and other proteins constantly synthesized throughout embryogenesis are useful references for these comparisons. Up to stages 26-27 only a few radioactive spots were present in the basic area of the gel. The most prominent spots have been identified as histones H3, H2b and H4 by their comigration with purified markers prepared from X. laevis erythrocytes and V8 protease mapping of the individual spots (data not shown). Ribosomal proteins are absent in these patterns, with the exception of S3, L17 and L31, which are present from early cleavage (spots A, C and D in Figure 4b), and L5, which appears at stages 7-9 (spot B in Figure 4b). Their identification, obtained by comigration with r-protein markers, was confirmed for S3 and L17 by V8 protease peptide mapping. The other radioactive proteins are not ribosomal and remain unidentified. At stages 26-27 a barely detectable r-protein pattern begins to appear, but a real full r-protein picture is observed only at stages 29-31 and later; the amount of r-protein mRNA per embryo increases less than 30% between these two stages. Thus the synthesized ribosomal proteins are detectable in embryos several hours after the appearance of their mRNA in the cytoplasm. Localization of r-Protein mRNA on Embryo Polysomal and Subpolysomal Fractions Since several hours intervene between the appearance of mRNA in the cytoplasm and its translation into proteins, the distribution of r-protein mRNA in subcellular fractions was investigated. Polysomal and subpolysomal fractions were prepared from stages 15, 26 and 31, and RNA was extracted as described in the Experimental Procedures. The RNA derived from
Cell 166
d
Figure
4. Two-Dimensional
Gel Electrophoresis
Analysis
of Proteins
Synthesized
during
Embryogenesis
Embryos of different stages were injected with 35S-methionine and incubated for 5-6 hr. Proteins were analyzed by two-dimensional etectrophoresis and fluorographed. Each gel was loaded with material corresponding to four embryos. The patterns of the most representative stages are shown. (b) Spots A, 6, C and D: the four “early” ribosomal proteins (S3, L.5. L17, L31) mentioned in the text. H: histones. (a) stages 2-5; (b) stages 79; (c) stages 14-l 6; (d) stages 26-27; (e) stages 29-31; (f) stag-e 41.
the two fractions of an equal number of staged embryos was run on agarose gels, blotted and hybridized to the radioactive probes. Figure 5 shows the hybridization pattern with the probe for r-protein Si mRNA. A band is present only on the subpolysomal fraction of stage 15. Later in development, a very faint band starts to appear on polysomes of stage 26, as the majority of mRNA for Sl is still on the subpolysomal
fraction. At stage 31 more than 50% of the mRNA is associated with polysomes. Thus mRNA for this ribosomal protein accumulates for several hours in subpolysomal particles and starts to be mobilized to polysomes around stage 26. The slower mobility of hybridization bands in the subpolysomal fractions of stages 15 and 25 is probably due to RNA overloading. In fact, at these stages the majority of ribosomes are
Ribosomal-Protein 167
Synthesis
in Xenopus
Development
present as monomers. This difference is no longer evident at stage 31, when a considerable amount of ribosomes is engaged in polysomes. Synthesis of Ribosomal Proteins and Accumulation of r-Protein mRNA in Anucleolate Mutants The anucleolate X. laevis mutants, which are deleted of rRNA genes, can survive up to the swimming stage with the use of maternal ribosomes (Brown and Gurdon, 1964). They provide the opportunity to study the coupling of r-protein and rRNA synthesis and to find out if regulation occurs at the transcriptional or translational level. Heterozygous X. laevis (one nucleolus) were mated and the progeny were screened as described in the Experimental Procedures. Pools of normal, heterozygous and anucleolate embryos were made according to the number of nucleoli present. Ten normal and ten anucleolate embryos were in--- 15 PS
26 Ps
31 Ps
Figure 5. Distribution of r-Protein mRNA polysomal Particles during Embryogenesis
on Polysomes
and Sub-
Cytoplasmic RNA extracted from polysomal (lanes p) and subpolysomal (lanes s) fractions from five embryos of stages 15, 26 and 31 was run on agarose gels, transferred to filters and hybridized to the probe pXom91 (ribosomal protein Si). Broadening of hybridization bands in lanes 15 s and 26 s could be due to overloading and/or to slight degradation of mRNA during preparation.
a
Figure
6. Synthesis
of Ribosomal
Proteins
in Anucleolate
jetted with 35S-methionine at stage 31 and incubated up to stage 35; during this period ribosomal proteins are already actively synthesized by normal embryos (see above). Figure 6 shows the autoradiographs of the two-dimensional pattern of acetic acid-extracted proteins from an equal number of labeled normal and mutant animals. In comparison with normal embryos (Figure 6a), the mutants do not synthesize most of the ribosomal proteins (Figure 6b) but only the four ribosomal proteins (S3, L17, L31 and L5) normally made at early stages (see above). The synthesis of histones and other nonribosomal proteins is unaffected; this is also true for those proteins that in normal embryos start to be synthesized during embryogenesis (some of these are indicated in Figure 6b). This suggests that the block of synthesis is specific for the ribosomal proteins. To investigate whether this specific inhibition of rprotein synthesis operates at the transcriptional or at the translational level, or both, we prepared cytoplasmic RNA from normal, heterozygous and anucleolate embryos at stages 26 and 35. The amount of total cytoplasmic RNA per embryo in the three different conditions is almost the same at stage 26, but it decreases by 20% in the heterozygous and by 50% in the anucleolate mutants at stage 35, as observed by Brown and Gurdon (1964). RNA corresponding to an equal number of animals was run on agarose gels and hybridized to probes for proteins Sl and L14. Figure 7 shows that mRNA for these ribosomal proteins was present in the mutants, although newly synthesized proteins were not found. At stage 26 mRNA accumulated in the three different genetic conditions, but at stage 35 the amount of mRNA in anucleolates decreased. (The first lane in Figure 7a was
b
Mutants
Ten anucleolate and ten normal sibling embryos were injected with ‘%-methionine at stage 31 and incubated up to stage 35. Experimental conditions were the same as described in Figure 4. Two-dimensional gels were loaded with material corresponding to two embryos. (a) Normal; (b) mutant. Spots A, B, C and D: the only ribosomal proteins synthesized by the anucleolate mutants: they are the same ribosomal proteins synthesized “early” by normal embryos (see Figure 4b). H: histones. Arrows: some nonribosomal proteins whose synthesis begins during embryogenesis and remains unaffected in anucleolates.
Cell 168
a Figure
7. Accumulation
b of r-Protein
mRNA
in Anucleolate
Cytoplasmic RNA from normal (lanes 2-nu). heterozygous nu) and anucleolate (lanes 0-nu) sibling embryos of stages was run on agarose gels, transferred to nitrocellulose hybridized to 32P-labeled pXom92 for ribosomal protein to “P-labeled pXom91 for ribosomal protein Sl (b). The (a) was not properly blotted, since it was at the edge of (b), the heterozygous sample of stage 26 is missing.
Mutants (lanes l26 and 35 filters and L14 (a) and first lane in the filter. In
not properly blotted, since it was at the edge of the filter.) The heterozygous RNA showed a normal hybridization in the two stages examined. Discussion The X. laevis oogenesis and early development represent two advantageous systems for the study, under physiological conditions, of two problems concerned with the regulation of ribosome biosynthesis: coordination of synthesis among ribosomal proteins, and coregulation with the synthesis of rRNA. Oocytes There are 10’” ribosomes accumulated in a single nondividing cell during oogenesis (Rosbash and Ford, 1974), a process that takes some months to complete. During this period the 18s and 28s RNA genes are amplified and a large subset of the 24,000 copies of 5s RNA genes are activated (Ford and Southern, 1973; Pelham et al., 1981). Ribosomal-protein genes, on the contrary, are not reiterated as far as somatic cells are concerned (Bozzoni et al., 1981). Since a coordination in the synthesis of rRNA and r-protein mRNAs was expected, we analyzed the accumulation of these RNA sequences in precisely staged oocytes. We found that the smallest oocytes from an adult ovary already contain mature r-protein mRNA at a level of about one fourth of the maximum, which is reached at late stage II (Dumont, 1972) before pigment deposition; beyond this stage the amount per cell is almost constant, with a reproducible reduction in fully mature oocytes. The same pattern was observed with the different probes tested. These mRNA sequences are stored in mRNP and are also engaged in protein synthesis, as we observed by analyzing the proteins produced by in vivo-labeled oocytes (Pier-
andrei-Amaldi and Beccari, 1980). The accumulation of r-protein mRNA precedes the maximal rate of ribosome production; according to Scheer (1973) in oocytes with a diameter of more than 400 pm the rate of accumulation of rRNA is still increasing, whereas at the same stage we find r-protein mRNA already at a steady state level. The pattern of accumulation of single mRNA sequences in X. laevis oocytes was analyzed by Golden et al. (1980). They tested mRNA sequences in different stages with cDNA probes copied from abundant oocyte mRNAs whose protein products were not known. All the nonmitochondrial RNA sequences tested followed the general pattern of poly(A)+ accumulation. They increased in amount from the very early stages, reaching a plateau at the onset of vitellogenesis. Our results indicate that r-protein mRNAs also follow a similar pattern. It was of interest to establish if the r-protein mRNAs are among the specific transcripts produced on the extended lampbrush chromosomes of X. laevis oocytes. We found that when lampbrush chromosomes are maximally extended (Dumont stage Ill), r-protein mRNAs have already reached a steady state level. According to Ficq (1968) however, there is some evidence of loop extension in very young oocytes, which we find actively accumulating r-protein mRNA. We must wait for a direct analysis of lampbrush chromosome transcripts by in situ hybridization to know if there is any r-protein mRNA synthesis on the loops. Embryos The ribosomes accumulated in the oocyte are utilized for protein synthesis during embryogenesis. This was clearly demonstrated in anucleolate mutants (Elsdale et al., 1958; Wallace, 1960) which, lacking genes for rRNA, can survive only up to early swimming stage (Brown and Gurdon, 1964). The normal embryo begins making the components of new ribosomes when still using the maternal ones. In fact, synthesis of rRNA was first detected at gastrulation, at which time nucleoli appear, and then it proceeds slowly. An increase of total RNA in the embryo occurs around the hatching stage (stage 35). At the end of embryogenesis (stage 42) the total RNA is about twice that of the unfertilized egg (Brown and Littna, 1964). We have followed the pattern of accumulation of rprotein mRNA by hybridization to specific recombinant DNA probes, which allows a quantitative evaluation of these sequences in the developing embryo. Newly made mRNA for the four ribosomal proteins tested (Ll , Li 4, Si and S19) begins to be detectable at gastrulation, and the amount per embryo increases rapidly during the period studied (up to stage 33). If we consider the concomitant increase of the number of cells per embryo, the amount of mRNA per cell goes up rapidly between gastrulation and mid-neurulation (stage 16) remaining constant afterwards. A
Ribosomal-Protein 169
Synthesis
in Xenopus
Development
dramatic increase of r-protein mRNA was also observed by in vitro translation of mRNA from staged embryos (Weiss et al., 1981). These results might reflect an activation of r-protein genes around the gastrula stage (1 O-l 1), together with the appearance of newly synthesized rRNA (Brown and Littna, 1964). However, a major change in r-protein mRNA turnover cannot be excluded. During early cleavage, some rprotein mRNA is present, but then it almost disappears (stages 7-9). The molecular weight indicates that it might belong to the short poly(A) mRNA population described by Sagata et al. (1980). These r-protein mRNA molecules are probably of maternal origin and are degraded during cleavage. We report that r-protein mRNA is not translated into proteins as soon as it appears, but accumulates into subpolysomal particles (mRNP) for several hours before becoming associated with polysomes. It is only at stage 26 that a small amount of r-protein mRNA can be detected on polysomes, most of it remaining on mRNP. Around this stage, barely detectable ribosomal proteins are synthesized in vivo. Within a few hours (stage 31). more than 50% of the mRNA is mobilized to polysomes, while an active synthesis of ribosomal proteins is carried on by the embryo. Four ribosomal proteins exhibit a peculiar behavior. In fact, S3, L17 and L31 are constantly synthesized from early cleavage and L5 from stages 7-9. They are reminiscent of the six peculiar ribosomal proteins that do not behave as most others in the yeast system (Gorenstein and Warner, 1976) of the small subset of ribosomal proteins that assemble in the ribosomes earlier than the others in the Drosophila embryo (Santon and Pellegrini, 1980) and of the anomalous behaviour of two rprotein mRNAs with respect to the others in regenerating rat liver (Faliks and Meyuhas, 1982). The “early” ribosomal proteins might have some significance in regulation of r-protein synthesis. Unfortunately, we do not have specific probes for them, for an analysis at the mRNA level. Several faintly visible ribosomal proteins were found by Weiss et al. (1981) among the products translated in vitro by stage 2 mRNA; these proteins were not detected again until stages subsequent to gastrulation. They could represent the in vitro translation products of the shorter mRNA sequences we find at very early stages. At stage 2, in contrast with in vitro translation, we do not find in vivo synthesis of ribosomal proteins other than those constantly synthesized. The concomitant appearance at gastrulation of rprotein mRNA (see above) and of newly synthesized rRNA (Brown and Littna, 1964) suggests that some common regulation may be involved in the expression of their genes. However, the onset of r-protein synthesis occurs around stages 29-31, in parallel with the significant increase of rRNA (Brown and Littna, 1964) and several hours after the appearance of their
mRNAs. This suggests that rRNA may play a role in controlling translation of r-protein mRNAs. The anucleolate mutants, which are deleted of genes for rRNA, seemed a suitable material to investigate these problems. The data presented here clearly show that the activation of the genes for ribosomal proteins occurs regularly in spite of the absence of rRNA synthesis, but later (stage 35) the amount of r-protein mRNA decreases. The synthesis of mRNA is not followed by a regular appearance of newly synthesized ribosomal proteins. This block is specific; in fact, other proteins are normally made in anucleolate embryos. In agreement with this, a normal attachment of rapidly labeled RNA to polysomes was reported by Gurdon and Ford (1967) in anucleolate mutants, and a regular synthesis of the nonribosomal proteins was described by Hallberg and Brown (1969). The only ribosomal proteins produced by the anucleolate mutants are the “early” ones (Sl, L17, L31 and L5). A first indication of this fact was obtained by Hallberg and Brown (1969). The step at which this block operates remains to be elucidated. It might operate at the level of mobilization of r-protein mRNA from mRNP to polysomes, as also observed by Geyer et al. (1982) for r-protein mRNA of growth-stimulated mouse fibroblasts; alternatively, ribosomal proteins might be synthesized but destroyed immediately because rRNA is not available, as suggested for other mammalian systems (Craig and Perry, 1971; Warner, 1977). The lack of rRNA or of ribosomal proteins, or both, seems to interfere, directly or indirectly, with the maintenance of r-protein mRNA accumulation. Experiments to elucidate this point are in progress. An overall picture of sequential events leading to the synthesis of new ribosomes in X. laevis developing embryos can be outlined. Ribosomal-protein mRNAs start to be synthesized at the same time as rRNA (stages 1 O-l 1) but independently of it, as shown in the anucleolate mutants. Newly made mRNAs are sequestered in mRNP; only at stages 26-30 do they move to polysomes and start to be actively translated when an appreciable amount of rRNA is accumulated. The presence of rRNA seems to be necessary for a normal translation of r-protein mRNAs. S3, L17, L31 and L5 escape the common mechanisms regulating the majority of ribosomal proteins. Experimental Biological
Procedures Materials
X. laevis frogs were purchased from Fish Hock, South Africa. Adult ovaries were disaggregated with 1 mg/ml collagenase (Boehringer Mannheim) at 25°C for 1 hr. Washed oocytes were staged by hand under the microscope according to the system of Dumont (1972). Embryos were obtained as described and were grown at 22°C in dechlorinated to the system of Nieuwkoop and Faber
Selection
of Anucleolate
One-nucleolate deletion, were
by Brown and Littna (1964)‘ water and staged according (1956).
Mutants
X. laevis frogs, heterozygous for the rRNA gene obtained from the Station de Zoologie Experimentale,
Cell 170
Universite de Geneve. The progeny of two heterozygotes were individually screened by analysis under the phase-contrast microscope of tissue fragments from the tailbud region, and were separated into different pools according to the number of nucleoli (Elsdale et al., 1958; Wallace, 1960). Cell Fractionation and RNA Preparation All glassware and solutions were sterile. RNA from 25 nonfractionated oocytes of each stage was extracted with SDS-phenol essentially as described by Ford et al. (1977). One hundred embryos from each stage were rinsed several times in 0.14 M NaCl and homogenized in 5 ml of 10 mM Tris (pH 7.4), 0.15 M KCI, 4 mM MgCI? and 0.005% Triton X-l 00. Nuclei were pelleted at 2000 rpm for 5 min and washed with 3 ml of 50 mM NaCl and 10 mM sodium acetate (pH 5). The two supernatants were pooled, and cytoplasmic RNA was extracted by a procedure similar to that described for RNA from oocytes. In some cases, a cytoplasmic fraction from 50 embryos was brought to a concentration of 1% deoxycholate, centrifuged at 11,000 rpm for 10 min and loaded onto 15%-50% sucrose gradients to separate polysomes from subpolysomal particles (Pierandrei-Amaldi et al., 1977). Pooled fractions were precipitated from the gradient with 3 volumes of ethanol at -20°C overnight. RNA was extracted from the precipitates as desorbed above. Poly(A)+ RNA was prepared as previously described (Pierandrei-Amaldi and Beccari. 1980). RNA gels RNA was separated on 0.8% agarose gels containing 20 mM morpholinepropane sulfonic acid (pH 7), 5 mM sodium acetate, 1 mM EDTA and 2.2 M formaldehyde in sterile water. Dry RNA precipitates were dissolved in lo-20 pl of the same solution containing 50% formamide, heated at 60°C for 5 min and cooled, 3 ~1 of 30% glycerol was added and the mixture was loaded onto the gels. Gels were run for about 3 hr at 150 V. recycling the buffer. At the end of the run, the gels were soaked for a few minutes in 20x SSC (3 M NaCI, 0.3 M trisodium citrate) and the RNA was transferred to nitrocellulose filters as described by Southern (1975) overnight in 1OX SSC. Dry filters were baked for 2 hr at 80°C. Hybridization Purified recombinant DNA inserts were ligated and radioactively labeled by nick translation (Manidtis et al.. 1976) to a specific activity on the order of 1 O8 cpm/pg. Hybridization to RNA bound to the filters and exposure of x-ray films were carried out as previously described (Bozzoni et al., 1981). The autoradiographs were quantitated optically with an EC model 910 recording densitometer: determinations on autoradiographs at different exposure times gave similar relative results. In vivo Labeling Embryos were dejelled with 1.5% cystein-HCI brought to pH 7-8, and were washed many times with sterile dechlorinated water. They were incubated whole or bisected in sterile water containing 150 Ag/ ml penicillin, 150 pg/ml streptomycin and 20 gCi ?S-methionine (New England Nuclear; spec. act. 1000 Ci/mmole), or preincubated with EDTA before labeling as described by Landesman and Gross (1968). or microinjected. Approximately 30 nl ?S-methionine (0.03 PCi) was injected by a glass microneedle in the dorsal area of the embryo. Ten embryos of each stage were injected and incubated in 0.5 ml sterile dechlorinated water containing antibiotics and 20 &i ?S-methionine. Incubation was carried out for 5-6 hr. Analysis of Synthesized Proteins After incubation, embryos were washed several times, and proteins were extracted, analyzed on two-dimensional gels and fluorographed as previously described (Pierandrei-Amaldi and Beccari, 1980). The only difference was that precipitation of extracted proteins with 7 volumes of acetone was preferred to lyophilization. Digestion of radioactive spots and of r-protein markers with Staphylococcus aureus V8 protease and peptide mapping analysis (Cleveland et al., 1977) were carried out as described by Bozzoni et al. (1981).
Acknowledgments We thank Prof. M. Fischberg and Dr. A Drain of the Station de Zoologie Experimentale. Department de Biologie Animale, Universite de Geneve, for providing the heterozygous X. laevis frogs. This research was partially supported by a grant from the lstituto PasteurFondazione Cenci Bolognetti. We are grateful to Mrs. Anna Sebastiano for typing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
February
2, 1982;
revised
May 14. 1982
References Amaldi. F., Beccari, E., Bozzoni, P. (1982). Nucleotide sequences for six Xenopus laevis ribosomal
I., Luo, Z. X. and Pierandrei-Amaldi, of cloned cDNA fragments specific proteins. Gene 17, 31 I-31 6.
Bachvarova. R. and Davidson, E. H. (1966). Nuclear activation onset of amphibian gastrulation. J. Exp. Zool. 163, 285-296.
at the
Bozzoni, I., Beccari, E., Luo, Z. X., Amaldi, F., Pierandrei-Amaldi, P. and Campioni, N. (1981). Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization. Nucl. Acid Res. 9, 1069-l 086. Brown, D. D. and Gurdon, J. 6. (1964). Absence synthesis in the anucleolated mutant of Xenopus Acad. Sci. USA 51, 139-146.
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Brown, D. D. and Littna, E. (1964). RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 8, 669-687. Cleveland, D. W., Fisher, S. G., Kirschner, M. W. and Laemmli. U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-l 106. Craig, N. and Perry, R. P. (1971). Persistent cytoplasmic synthesis of ribosomal proteins during the selective inhibition of ribosomal RNA synthesis. Nature New Biol. 229, 75-80. Dumont. J. N. (1972). of oocyte development 736, 153-180. Elsdale, reduces 643.
Oogenesis in Xenopus laevis (Daudin). Stages in laboratory maintained animals. J. Morphol.
T. R., Fischberg. nucleolar number
M. and Smith, S. (1958). A mutation that in Xenopus laevis. Exp. Cell Res. 74, 642-
Faliks, D. and Meyuhas. 0. (1982). Coordinate regulation of ribosomal protein mRNA in regenerating rat liver. Study with the corresponding mouse cloned cDNAs. Nucl. Acid Res. 10. 789-801. Ficq. A. (1968). Synthesis and detection Exp. Cell Res. 53, 691-693.
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Ford, P. J. and Southern, E. M. (1973). Different sequences for 5s RNA in kidney cells and ovaries of Xenopus laevis. Nature New Biol. 241, 7-l 2. Ford, P. J., Mathieson, T. and Rosbash, messenger RNA in ovaries of Xenopus 426.
M. (1977). Very long-lived laevis. Dev. Biol. 57, 417-
Geyer, P. K.. Meyuhas, O., Perry, R. P. and Johnson, L. F. (1982). Regulation of ribosomal protein messenger RNA content and translation in growth-stimulated mouse fibroblasts. Mol. Cell. Biol., in press. Golden, L., Schafer, individual PA+ RNAs 835-844.
U. and Rosbash. during oogenesis
M. (1980). Accumulation of of Xenopus laevis. Cell 22,
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Gurdon. J. B. and Ford, P. J. (1967). Attachment of rapidly labelled RNA to polysomes in the absence of ribosomal RNA synthesis during normal cell differentiation. Nature 216, 666-668. Hallberg. R. L. and Brown, D. D. (1969). Co-ordinated some ribosomal proteins and ribosomal RNA in embryos laevis. J. Mol. Biol. 46, 393-411.
synthesis of of Xenopus
Landesman, R. and Gross, P. R. (1968). Patterns of macromolecule synthesis during development of Xenopus laevis. I. Incorporation of radioactive precursor into dissociated embryos. Dev. Biol. 18, 571589.
Maniatis, T., Sim, G. K.. Efstratiadis, A. and Kafatos, F. C. (1976). Amplification and characterization of a ,&globin gene synthesized in vitro. Cell 8, 163-l 82. Nieuwkoop, P. D. and Faber, J. (1956). Normal laevis (Daudin). (Amsterdam: North-Holland).
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Nomura, M. and Post, L. E. (1980). Organization of ribosomal genes and regulation of their expression in Escherichia coli. In Ribosomes: Structure, Function and Genetics, G. Chambliss, G. R. Craven, J. Davies, K. Davis, L. Kahan and M. Nomura, eds. (Baltimore: University Park Press). pp. 671-691. Pelham, H. R. B., Wormington, W. M. and Brown, D. D. (1981). Related 5s RNA transcription factors in Xenopus oocytes and somatic cells. Proc. Nat. Acad. Sci. USA 78, 1760-l 764. Pierandrei-Amaldi, ribosomal proteins 603-611.
P. and Beccari E. (1980). Messenger RNA for in Xenopus laevis oocytes. Eur. J. Biochem. 706,
Pierandrei-Amaldi, P.. Felicetti, L. and Campioni, N. (1977). Flow of informational RNA from cytoplasmic poly(A)-containing particles to polyribosomes in Artemia salina cysts at early stages of development. Dev. Biol. 59, 49-61. Rosbash, M. and Ford, P. J. (1974). Polyadenyl-acid in Xenopus laevis oocytes. J. Mol. Biol. 85, 87-101.
containing
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Rosbash, M., Harris, P. K. W., Woolford, J. L., Jr., and Teem, J. L. (1981). The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell 24, 679-686. Sagata. N., Shiokawa, K. and Yamana, K. (1980). A study of the steady-state population of poly(A)+ RNA during early development of Xenopus laevis. Dev. Biol. 77, 431-448. Santon, J. B. and Pellegrini, M. (i 980). Expression of ribosomal proteins during Drosophila early development. Proc. Nat. Acad. Sci. USA 77, 5649-5653. Scheer. U. (1973). Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis. Dev. Biol. 30, 13-28. Southern, fragments 517. Wallace, Xenopus
E. M. (1975). Detection of specific separated by gel electrophoresis.
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H. (1960). The development of anucleolate laevis. J. Embryol. Exp. Morphol. 8, 405-413.
Warner, J. R. (1977). In the absence of ribosomal ribosomal proteins of HeLa cells are synthesized graded rapidly. J. Mol. Biol. 17 5, 315-333.
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Warner, J. R., Tushinski. R. J. and Wejksnora, P. J. (1980). Coordination of RNA and proteins in eukaryotic ribosome production. In Ribosomes: Structure, Function and Genetics, G. Chambliss, G. R. Craven, J. Davies, K. Davis, L. Kahan and M. Nomura, eds. (Baltimore: University Park Press), pp. 889-902. Weiss, Y. C., Vaslet, C. A. and Rosbash, mRNAs increase dramatically during Biol. 87, 330-339.
M. (1981). Ribosomal Xenopus development.
protein Dev.
71, August
1982,
Copyright
0 1982
by MIT
Expression of Ribosomal-Protein Genes in Xenopus laevis Development Paola Pierandrei-Amaldi and Nadia lstituto di Biologia Cellulare, C.N.R. Via Romagnosi 18/A 00196 Rome, Italy Elena Beccari, Irene Bozzoni and Francesco Amaldi Centro Acidi Nucleici, C.N.R. lstituto di Fisiologia Generale Universita di Roma 00185 Rome Italy
Campioni
Summary Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 24, become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs. Introduction The process of ribosome biosynthesis includes a number of reactions that together lead to the simultaneous and balanced production of the ribosomal components: the two large rRNAs, the 5s RNA and the ribosomal proteins. The mechanisms involved in the regulated expression of the ribosomal proteins and of the rRNA are fairly well understood in procaryotic systems (Nomura and Post, 1980) but are still not clear in eucaryotes. So far the regulation of ribosomalprotein (r-protein) and rRNA synthesis has been investigated in yeast (Gorenstein and Warner, 1976; Rosbash et al., 1981), in cultured animal cells (Craig and Perry, 1971; Warner, 1977; Geyer et al., 1982) and in regenerating rat liver (Faliks and Meyuhas,
1982). Although much has been learned, different results have been obtained in various systems and under different conditions, so that a general picture cannot be drawn (for review see Warner et al., 1980). We are interested in the Xenopus laevis system because it presents two physiological situations in which a change in the production of ribosomes occurs: oogenesis and early embryogenesis. In both cases a large amount of ribosomes is accumulated; the genes that encode the ribosomal proteins are therefore expected to be particularly active. An X. laevis oocyte cDNA bank was constructed with an mRNA fraction enriched for r-protein-coding capacity (Pierandrei-Amaldi and Beccari, 1980; Bozzoni et al., 1981). Clones carrying sequences for six different ribosomal proteins (Sl , S8, S19, Ll , L14 and L32) were selected by positive translation of mRNA specifically hybridized to the recombinant plasmids. We have used the recombinant clones as probes to monitor the behavior of specific r-protein mRNAs during oogenesis and embryogenesis. The pattern of accumulation of these mRNAs was followed and was related to the pattern of r-protein synthesis during embryogenesis. Anucleolate mutants were used to study the influence of rRNA on the expression of rprotein genes both at the level of mRNA production and at the level of r-protein synthesis. Results The sizes of the mRNA sequences specific for six ribosomal proteins were analyzed in X. laevis oocytes that had not yet completed vitellogenesis. Poly(A)+ RNA from these oocytes (Dumont stages I-III) has a good coding capacity for ribosomal proteins when tested in vitro. An enrichment for r-protein mRNA was obtained by selection of a 10-16s sucrose gradient fraction of the poly(A)+ RNA. It has been estimated that, all together, the 70 ribosomal proteins account for IO%-20% of the products coded in vitro by this mRNA fraction (Pierandrei-Amaldi and Beccari, 1980). Each mRNA sequence is thus present at a level of about 0.2%. Enriched poly(A)+ RNA was run in agarose gels, transferred to DBM paper or to nitrocellulose filters and hybridized to the six cloned cDNA probes for the different ribosomal proteins. The calibration was made with denatured RNAs as molecular weight standards: 28S, 18S, 5s and 4s RNAs from X. laevis and 9s RNA from rabbit reticulocyte; labeled denatured +X174 Hae Ill digestion fragments were also used as a reference. The average values of different experiments are reported in Table 1. The mRNA size is proportional to the protein length as determined on SDS-acrylamide gels (PierandreiAmaldi and Beccari, 1980). Accumulation of r-Protein mRNA during Oogenesis It was of interest to follow the amount of mRNA specific for ribosomal proteins during the course of oo-
Table 1. Length of Some r-Protein Previtellogenic X-laevis Oocytes
mRNA Sequences
in
Ribosomal Protein
Molecular Weight (Dalton&
Expected Length of mRNACoding Region (Nucleotides)
Ll
54,000
1,400
1,500
L14
20,800
540
750
pXom92
L32
8,800
230
550
pXom78
Sl
32,000
850
1,000
pXom91
S8
21,500
560
850
pXom62
s19
12.500
320
650
pXom69
Measured Length of mRNA (Nucleotides)
Recombinant Plasmid pXomlO2
Ribosomal proteins were numbered and tiieir molecular weight was calculated as described by Pierandrei-Amaldi and Beccari (1980). From the sequence of r-protein cDNAs (Amaldi et al., 19821, an average length of 115 amino acids was calculated. RNA length was determined on agarose gels; poly(A)+ RNA was transferred to DBM paper or nitrocellulose filters and probed with labeled cloned cDNA sequences.
genesis, in connection with the known features of rRNA synthesis. After collagenase digestion of the ovary walls, the free oocytes were washed several times to eliminate debris and were staged under a microscope into completely transparent oocytes, less than 250 pm in diameter (Dumont early I) (stage A); fully transparent, well visible nucleus, 250-300 pm (Dumont late I) (stage B); light yellow, round, nucleus not visible, 300-350 pm (Dumont II) (stage C); yellowbrown, starting pigmentation, 400-500 pm (Dumont Ill) (stage D); brown, half-size, about 600 pm (Dumont IV) (stage E); fully mature, 1.2-1.3 mm (Dumont VI) (stage F). Total RNA was extracted from 25 oocytes of each group, and amounts corresponding to ten oocytes were run on agarose gels under denaturing conditions and hybridized to the r-protein probes. The autoradiographs in Figure 1 show a reproducible increase of signal intensity from stages A to B to C and a stable level through stages D and E (Figures 1 a and 1 b). In fully matured oocytes the hybridizing signal was reduced. To rule out artifacts due to RNA overloading, we repeated the experiment with RNA corresponding to only two oocytes per lane, with identical results (Figure 1 c). Accumulation of r-Protein mRNA in the Embryo The accumulation of individual r-protein mRNA has been followed in early embryogenesis. To see the onset of r-protein mRNA synthesis, we analyzed several developmental stages from two blastomeres, up to the tailbud stage (stages 30-33). Cytoplasmic RNA was extracted from a fixed number of staged embryos (Nieuwkoop and Faber, 1956) generally 100, and the RNA corresponding to ten embryos was run on agarose gels under denaturing conditions. The amount of
ABCDEF Figure
1. Accumulation
of r-protein
mRNA during
Oogenesis.
Total RNA corresponding to ten (a and b) or two (c) oocytes of each stage was denatured, run on agarose gels and blotted on nitrocellulose filters. Filters were hybridized to “P-labeled pXom102 (ribosomal protein Ll) (a) or to 32P-labeled pXom92 (ribosomal protein L14) (b and c). (Lanes A-F) RNA isolated from corresponding oocyte stages (see text).
cytoplasmic RNA per embryo, mostly rRNA of maternal origin, was fairly constant in the period of time examined, about 3.5 pg. Only in the last two stages was a slight but reproducible increase observed. RNA was transferred from agarose to nitrocellulose membranes for hybridization with 3”P-nick-translated r-protein-specific probes. Figure 2 shows the pattern of accumulation of mRNA for ribosomal proteins L14 (Figure 2a) and Si (Figure 2b) as revealed by hybridization with the corresponding probes. mRNA starts to be detectable clearly at stages 10-l 1 (gastrula) and increases later on. A quantitative evaluation of the amount of mRNA present at different stages has been made by densitometric scanning of the autoradiographs. We have calculated the relative amount of mRNA per cell, taking into consideration the increasing number of cells per embryo (Figure 3). The amount of L14 and Sl mRNA increased rapidly between stages 10 and 16, and remained constant afterwards. A similar pattern was also observed for mRNAs of proteins Ll and S19 when hybridized to the corresponding probes (data not shown). In Figures 2a and 2b a faint hybridization band of slightly lower molecular weight can be seen at stages 2 and 5. This band almost disappears in the subsequent cleavage stages. The mRNA present at the beginning of embryogenesis probably represents residual maternal mRNA, and the slight difference in molecular weight may be due to shorter poly(A) tracts (Sagata et al., 1980). The probe for L14 mRNA was also hybridized to poly(A)+ RNA from embryos of different stages. Results similar to those for total RNA were obtained, including the slowmigrating band at early cleavage.
Ribosomal-Protein 165
Synthesis
in Xenopus
Development
a
oocyte
2
5
9
7
10
11
12
16
21
24
1
Figure
2. Accumulation
of r-Protein
mRNA
33 i
during
Embryogenesis
Cytoplasmic RNA derived from ten embryos of each stage was denatured, run on agarose gels and transferred to nitrocellulose filters as described in the Experimental Procedures. Filters were hybridized to ‘*P-labeled pXom92 (ribosomal protein L14) (a) or to pXom91 (ribosomal protein Si) (b). Numbers of lanes: embryo stages (Nieuwkoop and Faber, 1956). (Lane oocyte) cytoplasmic RNA from oocyte stages IV-VI (Dumont, 1972). In (b), stage 2 is missing.
Figure 3. Relative ogenesis
Amount
of r-Protein
mRNA
per Cell during
Embry-
The extent of hybridization of the r-protein (r-p) mRNA to the corresponding probes was estimated by densitometric tracing of the autoradiographs shown in Figure 2. (O- - -0) mRNA for ribosomal protein L14; (0- - -0) mRNA for ribosomal protein Sl The values have been normalized to the number of cells per embryo of the specific stage (Gurdon, 1974). Curves have been extrapolated to the last point. (A-A) Extraction yields of RNA from the different developmental stages.
Synthesis of Ribosomal Proteins in the Embryo To correlate the accumulation pattern of r-protein mRNAs with their translation, we followed the incorporation of 35S-methionine into ribosomal proteins during early embryogenesis. Embryos from early
cleavage to tadpole stages (stage 2-5, 7-9, 1 O-l 2, 14-16, 22-24, 26-27, 29-31 and 41) were labeled with 35S-methionine for 5-6 hr at 22°C. To circumvent the permeability barrier of the embryos, we tried different methods: bisection of embryos (Bachvarova and Davidson, 1966), EDTA treatment before incubation (Landesman and Gross, 1968) and injection with glass microneedles. The three methods gave the same qualitative results, but injection was chosen because the efficiency of incorporation was good and the embryos were not seriously damaged. Homogenates of incubated embryos were extracted with acetic acid and precipitated with acetone. Ribosomal proteins were then separated from total ceil proteins by two-dimensional electrophoresis (Gorenstein and Warner, 1976). Gels were loaded with extracts corresponding to four labeled embryos. Figure 4 shows examples of the fluorographs of the two-dimensional patterns of ribosomal proteins synthesized by embryos of different stages. Since, as mentioned, each gel was loaded with material from an equal number of embryos labeled under standard conditions, comparison of the patterns is possible. Moreover, histones (see below) and other proteins constantly synthesized throughout embryogenesis are useful references for these comparisons. Up to stages 26-27 only a few radioactive spots were present in the basic area of the gel. The most prominent spots have been identified as histones H3, H2b and H4 by their comigration with purified markers prepared from X. laevis erythrocytes and V8 protease mapping of the individual spots (data not shown). Ribosomal proteins are absent in these patterns, with the exception of S3, L17 and L31, which are present from early cleavage (spots A, C and D in Figure 4b), and L5, which appears at stages 7-9 (spot B in Figure 4b). Their identification, obtained by comigration with r-protein markers, was confirmed for S3 and L17 by V8 protease peptide mapping. The other radioactive proteins are not ribosomal and remain unidentified. At stages 26-27 a barely detectable r-protein pattern begins to appear, but a real full r-protein picture is observed only at stages 29-31 and later; the amount of r-protein mRNA per embryo increases less than 30% between these two stages. Thus the synthesized ribosomal proteins are detectable in embryos several hours after the appearance of their mRNA in the cytoplasm. Localization of r-Protein mRNA on Embryo Polysomal and Subpolysomal Fractions Since several hours intervene between the appearance of mRNA in the cytoplasm and its translation into proteins, the distribution of r-protein mRNA in subcellular fractions was investigated. Polysomal and subpolysomal fractions were prepared from stages 15, 26 and 31, and RNA was extracted as described in the Experimental Procedures. The RNA derived from
Cell 166
d
Figure
4. Two-Dimensional
Gel Electrophoresis
Analysis
of Proteins
Synthesized
during
Embryogenesis
Embryos of different stages were injected with 35S-methionine and incubated for 5-6 hr. Proteins were analyzed by two-dimensional etectrophoresis and fluorographed. Each gel was loaded with material corresponding to four embryos. The patterns of the most representative stages are shown. (b) Spots A, 6, C and D: the four “early” ribosomal proteins (S3, L.5. L17, L31) mentioned in the text. H: histones. (a) stages 2-5; (b) stages 79; (c) stages 14-l 6; (d) stages 26-27; (e) stages 29-31; (f) stag-e 41.
the two fractions of an equal number of staged embryos was run on agarose gels, blotted and hybridized to the radioactive probes. Figure 5 shows the hybridization pattern with the probe for r-protein Si mRNA. A band is present only on the subpolysomal fraction of stage 15. Later in development, a very faint band starts to appear on polysomes of stage 26, as the majority of mRNA for Sl is still on the subpolysomal
fraction. At stage 31 more than 50% of the mRNA is associated with polysomes. Thus mRNA for this ribosomal protein accumulates for several hours in subpolysomal particles and starts to be mobilized to polysomes around stage 26. The slower mobility of hybridization bands in the subpolysomal fractions of stages 15 and 25 is probably due to RNA overloading. In fact, at these stages the majority of ribosomes are
Ribosomal-Protein 167
Synthesis
in Xenopus
Development
present as monomers. This difference is no longer evident at stage 31, when a considerable amount of ribosomes is engaged in polysomes. Synthesis of Ribosomal Proteins and Accumulation of r-Protein mRNA in Anucleolate Mutants The anucleolate X. laevis mutants, which are deleted of rRNA genes, can survive up to the swimming stage with the use of maternal ribosomes (Brown and Gurdon, 1964). They provide the opportunity to study the coupling of r-protein and rRNA synthesis and to find out if regulation occurs at the transcriptional or translational level. Heterozygous X. laevis (one nucleolus) were mated and the progeny were screened as described in the Experimental Procedures. Pools of normal, heterozygous and anucleolate embryos were made according to the number of nucleoli present. Ten normal and ten anucleolate embryos were in--- 15 PS
26 Ps
31 Ps
Figure 5. Distribution of r-Protein mRNA polysomal Particles during Embryogenesis
on Polysomes
and Sub-
Cytoplasmic RNA extracted from polysomal (lanes p) and subpolysomal (lanes s) fractions from five embryos of stages 15, 26 and 31 was run on agarose gels, transferred to filters and hybridized to the probe pXom91 (ribosomal protein Si). Broadening of hybridization bands in lanes 15 s and 26 s could be due to overloading and/or to slight degradation of mRNA during preparation.
a
Figure
6. Synthesis
of Ribosomal
Proteins
in Anucleolate
jetted with 35S-methionine at stage 31 and incubated up to stage 35; during this period ribosomal proteins are already actively synthesized by normal embryos (see above). Figure 6 shows the autoradiographs of the two-dimensional pattern of acetic acid-extracted proteins from an equal number of labeled normal and mutant animals. In comparison with normal embryos (Figure 6a), the mutants do not synthesize most of the ribosomal proteins (Figure 6b) but only the four ribosomal proteins (S3, L17, L31 and L5) normally made at early stages (see above). The synthesis of histones and other nonribosomal proteins is unaffected; this is also true for those proteins that in normal embryos start to be synthesized during embryogenesis (some of these are indicated in Figure 6b). This suggests that the block of synthesis is specific for the ribosomal proteins. To investigate whether this specific inhibition of rprotein synthesis operates at the transcriptional or at the translational level, or both, we prepared cytoplasmic RNA from normal, heterozygous and anucleolate embryos at stages 26 and 35. The amount of total cytoplasmic RNA per embryo in the three different conditions is almost the same at stage 26, but it decreases by 20% in the heterozygous and by 50% in the anucleolate mutants at stage 35, as observed by Brown and Gurdon (1964). RNA corresponding to an equal number of animals was run on agarose gels and hybridized to probes for proteins Sl and L14. Figure 7 shows that mRNA for these ribosomal proteins was present in the mutants, although newly synthesized proteins were not found. At stage 26 mRNA accumulated in the three different genetic conditions, but at stage 35 the amount of mRNA in anucleolates decreased. (The first lane in Figure 7a was
b
Mutants
Ten anucleolate and ten normal sibling embryos were injected with ‘%-methionine at stage 31 and incubated up to stage 35. Experimental conditions were the same as described in Figure 4. Two-dimensional gels were loaded with material corresponding to two embryos. (a) Normal; (b) mutant. Spots A, B, C and D: the only ribosomal proteins synthesized by the anucleolate mutants: they are the same ribosomal proteins synthesized “early” by normal embryos (see Figure 4b). H: histones. Arrows: some nonribosomal proteins whose synthesis begins during embryogenesis and remains unaffected in anucleolates.
Cell 168
a Figure
7. Accumulation
b of r-Protein
mRNA
in Anucleolate
Cytoplasmic RNA from normal (lanes 2-nu). heterozygous nu) and anucleolate (lanes 0-nu) sibling embryos of stages was run on agarose gels, transferred to nitrocellulose hybridized to 32P-labeled pXom92 for ribosomal protein to “P-labeled pXom91 for ribosomal protein Sl (b). The (a) was not properly blotted, since it was at the edge of (b), the heterozygous sample of stage 26 is missing.
Mutants (lanes l26 and 35 filters and L14 (a) and first lane in the filter. In
not properly blotted, since it was at the edge of the filter.) The heterozygous RNA showed a normal hybridization in the two stages examined. Discussion The X. laevis oogenesis and early development represent two advantageous systems for the study, under physiological conditions, of two problems concerned with the regulation of ribosome biosynthesis: coordination of synthesis among ribosomal proteins, and coregulation with the synthesis of rRNA. Oocytes There are 10’” ribosomes accumulated in a single nondividing cell during oogenesis (Rosbash and Ford, 1974), a process that takes some months to complete. During this period the 18s and 28s RNA genes are amplified and a large subset of the 24,000 copies of 5s RNA genes are activated (Ford and Southern, 1973; Pelham et al., 1981). Ribosomal-protein genes, on the contrary, are not reiterated as far as somatic cells are concerned (Bozzoni et al., 1981). Since a coordination in the synthesis of rRNA and r-protein mRNAs was expected, we analyzed the accumulation of these RNA sequences in precisely staged oocytes. We found that the smallest oocytes from an adult ovary already contain mature r-protein mRNA at a level of about one fourth of the maximum, which is reached at late stage II (Dumont, 1972) before pigment deposition; beyond this stage the amount per cell is almost constant, with a reproducible reduction in fully mature oocytes. The same pattern was observed with the different probes tested. These mRNA sequences are stored in mRNP and are also engaged in protein synthesis, as we observed by analyzing the proteins produced by in vivo-labeled oocytes (Pier-
andrei-Amaldi and Beccari, 1980). The accumulation of r-protein mRNA precedes the maximal rate of ribosome production; according to Scheer (1973) in oocytes with a diameter of more than 400 pm the rate of accumulation of rRNA is still increasing, whereas at the same stage we find r-protein mRNA already at a steady state level. The pattern of accumulation of single mRNA sequences in X. laevis oocytes was analyzed by Golden et al. (1980). They tested mRNA sequences in different stages with cDNA probes copied from abundant oocyte mRNAs whose protein products were not known. All the nonmitochondrial RNA sequences tested followed the general pattern of poly(A)+ accumulation. They increased in amount from the very early stages, reaching a plateau at the onset of vitellogenesis. Our results indicate that r-protein mRNAs also follow a similar pattern. It was of interest to establish if the r-protein mRNAs are among the specific transcripts produced on the extended lampbrush chromosomes of X. laevis oocytes. We found that when lampbrush chromosomes are maximally extended (Dumont stage Ill), r-protein mRNAs have already reached a steady state level. According to Ficq (1968) however, there is some evidence of loop extension in very young oocytes, which we find actively accumulating r-protein mRNA. We must wait for a direct analysis of lampbrush chromosome transcripts by in situ hybridization to know if there is any r-protein mRNA synthesis on the loops. Embryos The ribosomes accumulated in the oocyte are utilized for protein synthesis during embryogenesis. This was clearly demonstrated in anucleolate mutants (Elsdale et al., 1958; Wallace, 1960) which, lacking genes for rRNA, can survive only up to early swimming stage (Brown and Gurdon, 1964). The normal embryo begins making the components of new ribosomes when still using the maternal ones. In fact, synthesis of rRNA was first detected at gastrulation, at which time nucleoli appear, and then it proceeds slowly. An increase of total RNA in the embryo occurs around the hatching stage (stage 35). At the end of embryogenesis (stage 42) the total RNA is about twice that of the unfertilized egg (Brown and Littna, 1964). We have followed the pattern of accumulation of rprotein mRNA by hybridization to specific recombinant DNA probes, which allows a quantitative evaluation of these sequences in the developing embryo. Newly made mRNA for the four ribosomal proteins tested (Ll , Li 4, Si and S19) begins to be detectable at gastrulation, and the amount per embryo increases rapidly during the period studied (up to stage 33). If we consider the concomitant increase of the number of cells per embryo, the amount of mRNA per cell goes up rapidly between gastrulation and mid-neurulation (stage 16) remaining constant afterwards. A
Ribosomal-Protein 169
Synthesis
in Xenopus
Development
dramatic increase of r-protein mRNA was also observed by in vitro translation of mRNA from staged embryos (Weiss et al., 1981). These results might reflect an activation of r-protein genes around the gastrula stage (1 O-l 1), together with the appearance of newly synthesized rRNA (Brown and Littna, 1964). However, a major change in r-protein mRNA turnover cannot be excluded. During early cleavage, some rprotein mRNA is present, but then it almost disappears (stages 7-9). The molecular weight indicates that it might belong to the short poly(A) mRNA population described by Sagata et al. (1980). These r-protein mRNA molecules are probably of maternal origin and are degraded during cleavage. We report that r-protein mRNA is not translated into proteins as soon as it appears, but accumulates into subpolysomal particles (mRNP) for several hours before becoming associated with polysomes. It is only at stage 26 that a small amount of r-protein mRNA can be detected on polysomes, most of it remaining on mRNP. Around this stage, barely detectable ribosomal proteins are synthesized in vivo. Within a few hours (stage 31). more than 50% of the mRNA is mobilized to polysomes, while an active synthesis of ribosomal proteins is carried on by the embryo. Four ribosomal proteins exhibit a peculiar behavior. In fact, S3, L17 and L31 are constantly synthesized from early cleavage and L5 from stages 7-9. They are reminiscent of the six peculiar ribosomal proteins that do not behave as most others in the yeast system (Gorenstein and Warner, 1976) of the small subset of ribosomal proteins that assemble in the ribosomes earlier than the others in the Drosophila embryo (Santon and Pellegrini, 1980) and of the anomalous behaviour of two rprotein mRNAs with respect to the others in regenerating rat liver (Faliks and Meyuhas, 1982). The “early” ribosomal proteins might have some significance in regulation of r-protein synthesis. Unfortunately, we do not have specific probes for them, for an analysis at the mRNA level. Several faintly visible ribosomal proteins were found by Weiss et al. (1981) among the products translated in vitro by stage 2 mRNA; these proteins were not detected again until stages subsequent to gastrulation. They could represent the in vitro translation products of the shorter mRNA sequences we find at very early stages. At stage 2, in contrast with in vitro translation, we do not find in vivo synthesis of ribosomal proteins other than those constantly synthesized. The concomitant appearance at gastrulation of rprotein mRNA (see above) and of newly synthesized rRNA (Brown and Littna, 1964) suggests that some common regulation may be involved in the expression of their genes. However, the onset of r-protein synthesis occurs around stages 29-31, in parallel with the significant increase of rRNA (Brown and Littna, 1964) and several hours after the appearance of their
mRNAs. This suggests that rRNA may play a role in controlling translation of r-protein mRNAs. The anucleolate mutants, which are deleted of genes for rRNA, seemed a suitable material to investigate these problems. The data presented here clearly show that the activation of the genes for ribosomal proteins occurs regularly in spite of the absence of rRNA synthesis, but later (stage 35) the amount of r-protein mRNA decreases. The synthesis of mRNA is not followed by a regular appearance of newly synthesized ribosomal proteins. This block is specific; in fact, other proteins are normally made in anucleolate embryos. In agreement with this, a normal attachment of rapidly labeled RNA to polysomes was reported by Gurdon and Ford (1967) in anucleolate mutants, and a regular synthesis of the nonribosomal proteins was described by Hallberg and Brown (1969). The only ribosomal proteins produced by the anucleolate mutants are the “early” ones (Sl, L17, L31 and L5). A first indication of this fact was obtained by Hallberg and Brown (1969). The step at which this block operates remains to be elucidated. It might operate at the level of mobilization of r-protein mRNA from mRNP to polysomes, as also observed by Geyer et al. (1982) for r-protein mRNA of growth-stimulated mouse fibroblasts; alternatively, ribosomal proteins might be synthesized but destroyed immediately because rRNA is not available, as suggested for other mammalian systems (Craig and Perry, 1971; Warner, 1977). The lack of rRNA or of ribosomal proteins, or both, seems to interfere, directly or indirectly, with the maintenance of r-protein mRNA accumulation. Experiments to elucidate this point are in progress. An overall picture of sequential events leading to the synthesis of new ribosomes in X. laevis developing embryos can be outlined. Ribosomal-protein mRNAs start to be synthesized at the same time as rRNA (stages 1 O-l 1) but independently of it, as shown in the anucleolate mutants. Newly made mRNAs are sequestered in mRNP; only at stages 26-30 do they move to polysomes and start to be actively translated when an appreciable amount of rRNA is accumulated. The presence of rRNA seems to be necessary for a normal translation of r-protein mRNAs. S3, L17, L31 and L5 escape the common mechanisms regulating the majority of ribosomal proteins. Experimental Biological
Procedures Materials
X. laevis frogs were purchased from Fish Hock, South Africa. Adult ovaries were disaggregated with 1 mg/ml collagenase (Boehringer Mannheim) at 25°C for 1 hr. Washed oocytes were staged by hand under the microscope according to the system of Dumont (1972). Embryos were obtained as described and were grown at 22°C in dechlorinated to the system of Nieuwkoop and Faber
Selection
of Anucleolate
One-nucleolate deletion, were
by Brown and Littna (1964)‘ water and staged according (1956).
Mutants
X. laevis frogs, heterozygous for the rRNA gene obtained from the Station de Zoologie Experimentale,
Cell 170
Universite de Geneve. The progeny of two heterozygotes were individually screened by analysis under the phase-contrast microscope of tissue fragments from the tailbud region, and were separated into different pools according to the number of nucleoli (Elsdale et al., 1958; Wallace, 1960). Cell Fractionation and RNA Preparation All glassware and solutions were sterile. RNA from 25 nonfractionated oocytes of each stage was extracted with SDS-phenol essentially as described by Ford et al. (1977). One hundred embryos from each stage were rinsed several times in 0.14 M NaCl and homogenized in 5 ml of 10 mM Tris (pH 7.4), 0.15 M KCI, 4 mM MgCI? and 0.005% Triton X-l 00. Nuclei were pelleted at 2000 rpm for 5 min and washed with 3 ml of 50 mM NaCl and 10 mM sodium acetate (pH 5). The two supernatants were pooled, and cytoplasmic RNA was extracted by a procedure similar to that described for RNA from oocytes. In some cases, a cytoplasmic fraction from 50 embryos was brought to a concentration of 1% deoxycholate, centrifuged at 11,000 rpm for 10 min and loaded onto 15%-50% sucrose gradients to separate polysomes from subpolysomal particles (Pierandrei-Amaldi et al., 1977). Pooled fractions were precipitated from the gradient with 3 volumes of ethanol at -20°C overnight. RNA was extracted from the precipitates as desorbed above. Poly(A)+ RNA was prepared as previously described (Pierandrei-Amaldi and Beccari. 1980). RNA gels RNA was separated on 0.8% agarose gels containing 20 mM morpholinepropane sulfonic acid (pH 7), 5 mM sodium acetate, 1 mM EDTA and 2.2 M formaldehyde in sterile water. Dry RNA precipitates were dissolved in lo-20 pl of the same solution containing 50% formamide, heated at 60°C for 5 min and cooled, 3 ~1 of 30% glycerol was added and the mixture was loaded onto the gels. Gels were run for about 3 hr at 150 V. recycling the buffer. At the end of the run, the gels were soaked for a few minutes in 20x SSC (3 M NaCI, 0.3 M trisodium citrate) and the RNA was transferred to nitrocellulose filters as described by Southern (1975) overnight in 1OX SSC. Dry filters were baked for 2 hr at 80°C. Hybridization Purified recombinant DNA inserts were ligated and radioactively labeled by nick translation (Manidtis et al.. 1976) to a specific activity on the order of 1 O8 cpm/pg. Hybridization to RNA bound to the filters and exposure of x-ray films were carried out as previously described (Bozzoni et al., 1981). The autoradiographs were quantitated optically with an EC model 910 recording densitometer: determinations on autoradiographs at different exposure times gave similar relative results. In vivo Labeling Embryos were dejelled with 1.5% cystein-HCI brought to pH 7-8, and were washed many times with sterile dechlorinated water. They were incubated whole or bisected in sterile water containing 150 Ag/ ml penicillin, 150 pg/ml streptomycin and 20 gCi ?S-methionine (New England Nuclear; spec. act. 1000 Ci/mmole), or preincubated with EDTA before labeling as described by Landesman and Gross (1968). or microinjected. Approximately 30 nl ?S-methionine (0.03 PCi) was injected by a glass microneedle in the dorsal area of the embryo. Ten embryos of each stage were injected and incubated in 0.5 ml sterile dechlorinated water containing antibiotics and 20 &i ?S-methionine. Incubation was carried out for 5-6 hr. Analysis of Synthesized Proteins After incubation, embryos were washed several times, and proteins were extracted, analyzed on two-dimensional gels and fluorographed as previously described (Pierandrei-Amaldi and Beccari, 1980). The only difference was that precipitation of extracted proteins with 7 volumes of acetone was preferred to lyophilization. Digestion of radioactive spots and of r-protein markers with Staphylococcus aureus V8 protease and peptide mapping analysis (Cleveland et al., 1977) were carried out as described by Bozzoni et al. (1981).
Acknowledgments We thank Prof. M. Fischberg and Dr. A Drain of the Station de Zoologie Experimentale. Department de Biologie Animale, Universite de Geneve, for providing the heterozygous X. laevis frogs. This research was partially supported by a grant from the lstituto PasteurFondazione Cenci Bolognetti. We are grateful to Mrs. Anna Sebastiano for typing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
February
2, 1982;
revised
May 14. 1982
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