Messenger ribonucleoprotein particles in developing sea urchin embryos

DEVELOPMENTAL

BIOLOGY

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Messenger

Ribonucleoprotein Particles in Developing Sea Urchin Embryos ELIHU

Program

(19%)

M. YOUNG’ AND RUDOLF A. RAFF

in Molecular, Cellular and Developmental Biology and Department Indiana University, Bloomington, Indiana 47405 Received October 16, 1978; accepted in revised form February

of Biology,

27, 1979

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)‘mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm“ in CslSOI. Poly(A)‘mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm’, while EDTA caused a shift in density to 1.55 g/cm”, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55-1.66 g/cmJ, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [‘Hluridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs,SO, gradients at a density of 1.60-1.66 g/cm”, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10“; protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.

onic transcription (Galau et al., 1977; Rough-Evans et al.7 1977). The use of actinomycin D (Gross and Cousineau, 1963; Greenhouse et al., 1971; Sargent and Raff, 1976) has made it possible to observe the post-transcriptional modification and translation of stored mRNAs in embryos in the absence of transcription since such embryos continue to develop and reach the hatched blastula stage before development ceases. Newly synthesized mRNA is easily observed by incorporation of isotopically labeled RNA precursors since mRNA synthesis predominates in embryos until the gastrula stage. We have previously demonstrated that

INTRODUCTION

During the early development of sea urchin embryos, two sources of messenger RNA are utilized. The initial rise in protein synthesis following fertilization is independent of concomitant transcription and results from the mobilization of mRNA sequences stored in the egg. Embryonic transcription begins at the B- to l&cell stage (Wilt, 1970) and new transcripts gradually replace the oogenetic mRNA until by the gastrula stage essentially all functioning mRNA in the embryo represents embry’ Present address: Sidney Farber Cancer Institute, Harvard Medical School, Boston, Mass. 02115. 24 0012-1606/79/090024-17$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Sea Urchin Embryo mRNPs

both the poly(A)+ and histone mRNAs of unfertilized eggs are contained as masked mRNA within nontranslatable ribonucleoprotein structures (mRNPs) (Kaumeyer et al., 1978; Jenkins et al., 1978; Raff, 1979; Shepard, 1977). These particles are free in the cytoplasm of eggs: Histone mRNPs sediment at 20-40 S and the poly(A)+mRNPs at 40-80 S. Both classes have densities in C&SO4 characteristic of RNA-protein complexes (DeFilippes, 1965; Hamilton, 1971; Wilt et al., 1973). Following fertilization, a portion of the oogenetic mRNA is “unmasked” by a mechanism as yet unelucidated and enters polysomes to support the initial rise in protein synthesis (Humphreys, 1971). However, a considerable fraction of the mass and of the sequence complexity of oogenetic mRNA remains unassociated with ribosomes (Dolecki et al., 1977; Hough-Evans et al., 1977). Embryonic transcription becomes significant by the 16-cell stage with histone mRNA accounting for about 60% of the mRNA produced during cleavage (Nemer, 1975). Newly transcribed mRNA exits the nucleus rapidly and is predominantly present in polysomes though some is found in structures not associated with ribosomes (Infante and Nemer, 1968; Dworkin and Infante, 1976). Thus four classes of presumptive mRNAcontaining RNPs exist within sea urchin embryos. We have investigated the RNP structure of representatives of these four classes: (i) oogenetic poly(A)‘mRNA released from polysomes, (ii) oogenetic poly(A)+mRNA in subpolysomal structures, (iii) newly transcribed mRNA released from polysomes, and (iv) newly transcribed mRNA in subpolysomal structures. Oogenetic mRNA from the free cytoplasmic pool or released from polysomes is contained in stable protein-rich mRNPs. Newly transcribed RNA, on the contrary, whether released from polysomes or found free in the cytoplasm exists in mRNPs with

very little protein. Our results are consistent with the hypothesis that there are differences in the regulation of translation of oogenetic poly(A)+RNA and the newly transcribed mRNA. While unmasking of oogenetic mRNA is a gradual process, the bulk of the newly transcribed mRNA in early development is histone mRNA which is rapidly translated upon entering the cytoplasm. MATERIALS

AND

METHODS

Materials Sea urchins (Stronglylocentrotus purpuratus) were purchased from Controlled Environments, Bellevue, Washington, and Pacific Biomarine, Venice, California. Actinomycin D (Grade I), heparin (Grade I), diethylpyrocarbonate, RNase A Type II-A, penicillin G, and streptomycin sulfate were all purchased from Sigma Chemical Company. Cs2S0, (optical grade) was bought from Kawecki-Berylco Industries, Inc. [5,63H]Uridine (specific activity 34-47 Ci/ mmole) and [5,6-3H]poly(U) (4.2-4.6 Ci/ mmole) were bought from New England Nuclear Corporation. Proteinase K was obtained from EM Labs. Oligo(dT)-cellulose (T3) was purchased from Collaborative Research Inc. Puromycin dihydrochloride (A Grade) was obtained from Calbiochem. All solutions were treated with diethylpyrocarbonate and autoclaved for 10 min. All glassware was baked prior to use.

Culturing and Labeling of Embryos Gametes were obtained by intracoelomic injection of 0.5 M KCI, and eggs were washed with Millipore-filtered sea water (MPFSW) containing 40 pg/ml penicillin G and streptomycin sulfate. For culturing embryos, eggs were fertilized with a dilute sperm suspension and swirled continuously at 18°C in a 1% suspension. Only cultures exhibiting at least 95% normal cleavage were used.

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To label RNA transcribed during cleavage, embryos were cultured for 7 hr (approximately 128-cell stage), concentrated to a 10% suspension, and labeled 90 min with [5,6-3H]uridine at 20 &i/ml. This amount of time is sufficient to allow polysomes to reform (Farquhar and McCarthy, 1973). For the study of oogenetic mRNA, eggs were pretreated 30 min prior to fertilization with 20 pg/ml actinomycin D and cultured in the dark in the continued presence of actinomycin (Greenhouse et al., 1971; Sargent and Raff, 1976).

VOLUME 72, 1979

were used to release structures containing mRNA from polysomes. In the first, resuspended polysomes were brought to 30 mM EDTA and incubated on ice 10 min. In the second method (Blobel, 1971), polysomes were resuspended in 0.5 M KCl, 5 mJ4 magnesium acetate, 0.05 M triethanolamine W-I 7.5), containing 2 mJ4 puromycin (which had been adjusted to pH 7) and 0.1% Triton X-100, then incubated 5 min at 4°C and 15 min at 37°C. The incubated fractions (which consisted of released mRNAcontaining structures and ribosomal subunits) were layered over 5-20% (w/v) linear Isolation of Polysomes and Subpolysomal sucrose gradients. Pelleting of polysomes Structures Containing mRNA had no effect upon release of mRNPs. The All procedures were carried out at 4°C. pattern obtained with polysomes that had At the desired times, embryos were har- been pelleted and resuspended prior to revested by centrifugation at 200g for 1 min. lease was indistinguishable from that obEmbryos were washed once in calcium- tained from unpelleted polysomes. magnesium-free sea water and twice with The distribution across gradients of TKM buffer (0.35 M KCl, 5 mM magnesium structures containing newly transcribed acetate, 0.05 M triethanolamine, pH 7.6). RNA was monitored by precipitating aliEmbryos were homogenized with a Dounce quots from each fraction with 15% TCA. homogenizer (using the tight-fitting pestle) Samples were filtered and counted in a toluene-based scintillation fluid. Polyin at least 7 vol of TKM made 0.1% Triton X-100,500 pg/ml heparin. The homogenate (A)+RNA was detected by hybridization of was centrifuged at 12,000g for 10 min and [3H]poly(U) as described by Kaumeyer et the supernatant layered onto 15-30% (w/v) al. (1978) and outlined below. linear sucrose gradients made in TKM buffer. Gradients were centrifuged at Detection of Poly(A) Aliquots of each fraction were precipi9O,OOOg,,for 2 hr. To insure that mRNA was undegraded, polysomes that did not tated with 2 vol of ethanol, pelleted, and appear to be intact (as judged by AZ& were resuspended in TSE-SDS (0.025 M Tris, discarded. For the isolation of subpolyso- 0.1 M NaCl, 5 mM EDTA, and 0.5% SDS, mal structures containing mRNA, fractions pH 7.4). To each fraction was added 0.2 ml sedimenting at 80 S and slower were recen- of TSE-SDS containing 5 mg/ml proteintrifuged on 20-40% (w/v) linear sucrose gra- ase K. Samples were incubated at 37°C for dients as indicated in Fig. 1. 60 min and precipitated with ethanol at -20°C. Precipitates were pelleted and reRelease of Structures Containing mRNA suspended in 2~ SSC (0.3 M NaCl, 0.03 M from Polysomes sodium citrate), and [3H]poly(U) was added Fractions containing polysomes were in 10 ~1 of 2~ SSC. Samples were heated at pooled and pelleted by centrifugation at 60°C for 5 min and hybridized for 30 min at 160,OOOgfor 10 hr. The supernatant was 37°C. After cooling to 3O”C, fractions were aspirated, polysomes were resuspended in mixed with 0.5 vol of 0.84 M NaCl, 0.03 M TKM buffer, and insoluble material was MgC12, 0.03 M Tris (pH 7.4) containing 15 removed by centrifugation. Two techniques ,ug/ml RNase A and digested at 30°C for 60

YOUNG AND RAFF

Sea Urchin Embryo mRNPs

min. RNase-resistant material was precipitated with 15%TCA. Samples were filtered and counted. As reported by Kaumeyer et al. (1978) detection of poly(A) is linear in [3H]poly(U) excess.

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tRNA never reaches the density expected of pure RNA. We therefore centrifuged CssSOl gradients for 40 hr or longer in all experiments. Gradients were fractionated either by puncturing the bottom of the tube with a 21-gauge needle and collecting 20drop fractions, or by pumping the gradients Extraction and Sizing of RNA out from the bottom at 1 ml/min while Fractions corresponding to selected S- collecting fractions every 15 sec. Density of value ranges were pooled from sucrose gra- every third fraction was determined by redient fractionations of mRNA-containing fractive index. structures, mixed with Escherichia coli For determination of the position of larRNA carrier, and precipitated with 2 vol beled RNA in the density gradients, samof ethanol. Samples were resuspended in ples were precipitated with TCA, filtered, 0.1 MTris, 0.5% SDS (pH 9.0) and extracted and counted. Recovery of CszSOssamples with water-saturated phenol:chloroform: for [3H]poly(U) hybridization was accordisoamyl alcohol (50:48:2), followed by chlo- ing to Kaumeyer et al. (1978). Each fraction roforrmisoamyl alcohol (96:4). The aqueous was digested for 1 hr with 1 ml of TSE phase was brought to 0.2 M potassium ace- (without SDS) containing 500 pg proteinase tate and RNA was precipitated at -20°C K at 37’C. To each fraction, 80 pg of E. coli with ethanol. RNA was pelleted, resus- rRNA (which shows no detectable [3H]pended in SDS buffer (0.1 M NaCl, 1 m&I poly(U) binding) was added as carrier and EDTA, 0.5% SDS, 0.01 M Tris, pH 7.5), and the fractions precipitated with 1 ml of 5 M heated at 60°C for 10 min. Samples were LiCI. After precipitation overnight at 4”C, layered on 15-30% (w/v) linear sucrose gra- samples were pelleted, dissolved in 2~ SSC, dients made in SDS buffer. Gradients were and hybridized to [3H]poly(U). Recovery of centrifuged at 196,000g av for 6.5 hr at hybridizable poly(A) was 70% (Kaumeyer 23°C. Labeled newly synthesized RNA was et al., 1978). precipitated with 15% TCA and counted on filters. Poly(A)+RNA samples were precip- Determination of the Poly(A) Content of itated with ethanol and hybridized to Newly Transcribed RNA [3H]poly(U) as described. RNA labeled with r3H]uridine and released from polysomes by EDTA was fracFormaldehyde Fixation and Cs2S04 Dentionated as described. Each fraction was sity Gradients precipitated with 2 vol of ethanol, dissolved Samples taken from sucrose gradients in TSE-SDS, and digested with proteinase were fixed from 4 to 24 hr at 4°C brought K as outlined above. Samples were pelleted to a final concentration of 4% formaldehyde and dissolved in 0.5 M NaCl, 0.01 M Tris (adjusted to pH 7), then loaded on pre- (pH 7.5), with 0.1% SDS. Each fraction was formed CsS04 gradients ranging in density passed over an oligo(dT)-cellulose (T3) colfrom 1.2 to 1.7 g/cm” made in 0.5 M triethumn equilibrated with the same high-salt anolamine (pH 7.2). Centrifugation was in buffer. After thorough washing, bound maeither Beckman SW 50-L or SW 65 rotor at terial was eluted with 0.01 M Tris (pH 7.5) 125,000g. We have found that high molec- containing 0.1% SDS. Carrier bovine serum ular weight RNA (21 S) will reach equilib- albumin was added to bound and unbound rium after 16 hr of centrifugation in pre- fractions and the samples were precipitated formed gradients while 9 S histone mRNA with TCA and counted. Percentage of larequires 40 hr to reach equilibrium, and beled RNA that bound to the column as

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VOLUME 72. 1979

well as the percentage recovery was determined.

Electrophoresis Gels

of RNA on Formamide

Formamide gels were prepared according to the procedure of Pinder et al. (1974). Extracted RNA was dried under Nz, dissolved in formamide buffer (0.02 M barbital, 10% sucrose, in deionized formamide), and heated at 37°C for 5 min prior to loading. Electrophoresis was at 5 mA/gel for 1 hr. Gels were stained for 3 hr with 0.1% pyronine Y in 0.5% acetic acid and 1 rmVl citric acid. Destaining was with 10% acetic acid. Gels were sliced and dissolved by incubating in 0.3 ml of 15% HzO:! at 50°C overnight. Slices were counted in 2 ml of Scintisol:toluene (1:l) and 10 ml of toluenebased scintillation fluid. RESULTS

Oogenetic Poly(A)+mRNAs and Newly Transcribed mRNAs in Subpolysomal mRNPs of Embryos While recruitment of oogenetic mRNA into functioning polysomes begins shortly following fertilization, a significant pool of oogenetic mRNA remains in the cytoplasm (Hough-Evans et al., 1977). Since recruitment is gradual (Dolecki et al., 1977) it appears likely that the free cytoplasmic mRNA remains in mRNPs similar or identical to those of the unfertilized egg. If this hypothesis is correct, the structure of remaining cytoplasmic mRNPs following fertilization and initiation of translation should be the same as in the unfertilized egg. The sedimentation behavior of subpolysomal mRNPs containing oogenetic poly(A)‘mRNAs is compared with that of subpolysomal mRNPs containing newly synthesized mRNAs in Fig. 1. Embryos cultured in the presence of 20 pg/ml of actinomycin D were allowed to develop for 12 hr to the early blastula stage

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FRACTION NUMBER FIG. 1. Distribution of oogenetic poly(A)‘RNA and newly transcribed RNA in subpolysomal fractions of embryos. Fractions sedimenting slower than 80 S on sucrose gradients of embryos either labeled with [“Hluridine or cultured in actinomycin D were recentrifuged on 20-40s (w/v) linear sucrose gradients (in TKM buffer) in an SW 27 rotor at 26,006 rpm for 17 hr at 4’C. Distribution of labeled RNA was monitored by TCA precipitation while poly(A)‘RNA was detected by hybridization to [“H]poly(U). 0, Newly transcribed [“HI-labeled RNA; X, oogenetic poly(A)‘RNA; p, optical density. Direction of sedimentation in this and all subsequent figures is from right to left. S values of mRNPs were estimated from S values of ribosomal subunits (Infante and Krauss, 1971; D. E. Leister, unpublished data).

(about 500 cells), homogenized, and fractionated to yield polysomes and subpolysomal fractions as described under Materials and Methods. The subpolysomal fractions (sedimenting more slowly than 80 S) were pooled and recentrifuged on a second set of sucrose gradients under conditions in which monosomes traverse 80% of the length of the gradient. Oogenetic poly(A)‘mRNA was detected in fractions recovered from this gradient by hybridization as described under Materials and Methods. The optical density trace shows the position of ribosomal subunits in the gradient. The distribution [“HI-labeled mRNAcontaining structures from the subpolysoma1 (80 S) fractions of embryos cultured for

YOUNG AND RAFF

Sea Urchin Embryo mRNPs

9 hr are also plotted in Fig. 1. Embryos were labeled with C3H]uridine for 90 min (to the 250-cell stage), homogenized, and fractionated as described under Materials and Methods. Fractions sedimenting more slowly than 80 S were collected and centrifuged on a second set of gradients under conditions in which monosomes travel through 80% of the gradient. Oogenetic poly(A)‘RNA is present in RNPs sedimenting from 35 to 80 S with a peak of 60-70 S. This S-value distribution for oogenetic mRNA-containing structures free in the cytoplasm of embryos is identical to that we previously observed for the cytoplasmic mRNPs of unfertilized eggs (Kaumeyer et al., 1978). The sedimentation pattern for particles containing newly synthesized mRNAs is more complex. There is a broad distribution of labeled RNA from 40 to 80 S with a modal sedimentation of about 65 S, and a larger peak of material at the top of the gradient. This pattern was consistently reproducable. The fractions containing oogenetic or newly transcribed RNA were fixed and examined by isopycnic centrifugation on

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Cs2S04gradients. The results are shown in Figs. 2A and 3. The buoyant density, in Cs2S04,of the 60-70 S subpolysomal particles containing oogenetic poly(A)‘mRNA ranged from 1.45 to 1.48 g/cm”, with a peak at 1.46 g/cm” (Fig. 2A). This is identical to the density of 1.46 g/cm3 observed for RNPs present in the unfertilized egg, and indicates that as in eggs, the oogenetic mRNA in the cytoplasm of embryos is present in mRNPs. The buoyant densities of mRNPs containing newly synthesized mRNAs were found to be distinctly different from the densities of oogenetic mRNPs. Labeled material from the 65 and 40 S fractions had a density range of 1.55-1.66 g/cm” (Figs. 3A and B) indicative of a low protein content (maximum of 10%). Figure 3C shows the density of material from the top of the gradient (fractions 8-10) to be 1.45 g/cm” which is suggestive of typical mRNA-protein complexes. However, we have extracted RNA from the top of the gradient and electrophoresed it on formamide gels, which give a clear separation of 4 S tRNA and 5 S rRNA from 9 S histone mRNA.

FRACTION NUMBER FIG. 2. Buoyant density of subpolysomal structures containing oogenetic poly(A)+RNA of embryos. Panel A shows the density of subpolysomal particles sedimenting at 60-70 S which contain the oogenetic poly(A)‘RNA, fixed with formaldehyde and centrifuged on C&O, gradients. RNA was recovered and hybridized to [‘HIpoly(U) as described. Panel B shows the result of making the 70 S RNPs to 30 mM EDTA prior to fixation. Panel C shows the density of the 55 S poly(A)‘mRNP released from polysomes by IDTA. Background [no poly(A)] is approximately 100 cpm, and has not been subtracted.

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FIG. 3. Buoyant density of subpolysomal structures containing newly transcribed RNA, labeled with [‘HIuridine. Subpolysomal RNA labeled with [3H]uridine sedimenting at 65 S (A) and 40 S (B) as well as material remaining at the top of the gradient (C) was fixed with formaldehyde and centrifuged on preformed Cs2S04 gradients at 125,OOOg for 2 days at 4°C. Following fractionation samples were precipitated with TCA, filtered, and counted.

Our results (data not shown) indicate that little mRNA is present, and that 93% of the [3H]uridine-labeled RNA is 4 and 5 S RNA. Because of their small size the tRNAs and 5 S rRNAs do not band at the density expected of free RNA even after >48 hr of centrifugation in Cs$S04 gradients. Thus the low density of RNA-containing fractions from the top of the gradient is indicative not of mRNPs, but of small, 4 and 5 S, RNAs. In contrast, a similar experiment with polysomal RNA shows that 90% of the labeled low molecular weight RNA (less than 15 S) is histone mRNA and only 10% is 4 and 5 S RNA.

Oogenetic Poly(A)+mRNA mRNPs

in Polysomal

To determine if oogenetic mRNA recruited into polysomes continues to be associated with protein in a stable mRNP, eggs were pretreated with actinomycin D 30 min prior to fertilization and the embryos allowed to develop in the presence of actinomycin to late cleavage-early blastula

stage. Polysomes were isolated and mRNA released as described. Released mRNPs and ribosomal subunits were centrifuged on sucrose gradients and aliquots of each fraction were assayed for poly(A)+mRNA. Particles containing oogenetic poly(A)+mRNA were detected by hybridization of [3H]poly(U). The poly(A)‘mRNA released from polysomes (Fig. 4) sediments as a broad peak from 25 to 65 S with a modal sedimentation of 55 S, after release from polysomes by EDTA. The extent of contamination of the polysomal fraction by mRNA-containing structures not attached to polysomes was estimated by EDTA disruption of polysomes. Heterogeneous structures containing poly(A)‘mRNA sedimenting at 65 S were found to contain no more than 10% of the poly(A)+mRNA found in polysomes and so were negligible in this study (data not shown). We have investigated the stability of free cytoplasmic mRNPs to exposure to 30 m&f EDTA since 30 mM EDTA is commonly used to release mRNA or mRNPs from polysomes, and has been so used in this

YOUNG AND RAFF

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31

fuged to equilibrium on C&SO4 gradients. [3H]Poly(U) was hybridized to Cs804 fractions as described under Materials and Methods. Puromycin release (Fig. 5A) yielded mRNPs with densities ranging from 1.42 to 1.52 g/cm3 and a peak at 1.46 g/cm3. The peak fraction thus represents a particle that is approximately 50% protein by 0 25 weight (Hamilton, 1971). EDTA release (Fig. 5B) which is known to remove proteins from ribosomes (Spitnik-Elson and Atsmon, 1969; Kumar and Lindberg, 1972) caused a shift in density to 1.46-1.60 g/cm3, 123456789 with a peak of 1.55 g/cm3. Particles released FRACTION NUMBER with puromycin and subsequently exposed to EDTA (Fig. 5C) showed an even more FIG. 4. Sedimentation of oogenetic poly(A)‘mRNPs released from polysomes by EDTA. Poly- pronounced shift in density to 1.60-1.66 g/ somes isolated from embryos cultured in actinomycm3, with a peak at 1.62 g/cm3. These retin D were pelleted, resuspended in TKM buffer, and sults are comparable to those of Kumar and made 30 mM EDTA. Released material was centrifuged on 5-20s (w/v) linear sucrose gradients (in Lindberg (1972), who showed that EDTA removes proteins from RNPs. TKM). Centrifugation was in an SW 41 rotor at 40,000 treatment rpm for 2 hr at 4°C. Gradients were fractionated and The density of the puromycin-released paraliquots of each fraction were hybridized to r3H]- ticles is similar to those reported by Kaupoly(U) as described under Materials and Methods. meyer et al. (1978) for free RNPs isolated -, Azw; 0, [3H]poly(U) hybridized. from the cytoplasm of unfertilized eggs in study. As shown in Fig. 2B treatment of the presence of 0.35 M KC1 and 5 mM Mg” or 5 m&I EDTA. these particles with 30 m&I EDTA resulted in about half the mass of particles assuming Newly Transcribed RNA in Polysomes a density of 1.60 g/cm3 which is consistent with a significant loss of protein. These At least 90% of the labeled RNA in the polysome region is sensitive to EDTA. MesEDTA-treated free cytoplasmic mRNPs have a similar density profile to polysomal senger RNA released from polysomes with particles containing oogenetic mRNA and either EDTA or puromycin was centrifuged released by EDTA (Fig. 2C), with major on a second set of sucrose gradients. Figure peaks at 1.55 and 1.43 g/cm3. 6 illustrates a typical pattern of structures An alternative method of releasing oog- containing labeled RNA released from enetic mRNA-containing structures from polysomes by EDTA. The optical density polysomes was by use of puromycin as de- trace represents ribosomal subunits. Two scribed under Materials and Methods. broad peaks of radioactivity were found, Poly(A)‘mRNPs released from polysomes one ranging from 25 to 35 S with a modal with puromycin sedimented with an S- sedimentation of 30 S and the other from value distribution similar to that of EDTA10 to 20 S with a modal sedimentation of released material. That oogenetic mRNA is 15 S. Similar experiments (polysome isolareleased from polysomes as mRNPs by purtion and EDTA release) were performed omycin or EDTA was confirmed by buoyunder a variety of salt conditions ranging ant density analysis. from 0.1 to 0.5 M NaCl as well as 0.25 to 0.5 The 55 S fraction from sucrose gradients M KCl. While there were some differences was fixed with formaldehyde and centriin the sedimentation of both ribosomal sub-

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FIG. 5. Buoyant density of structures containing oogenetic poly(A)‘mRNA released from polysomes. Polysomes were isolated from embryos cultured in actinomycin D and the mRNA was released by puromycin or EDTA as described under Materials and Methods. Poly(A)‘mRNA in particles with a modal sedimentation of 55 S were fixed with formaldehyde and centrifuged on C&O, gradients for 24 hr. RNA was recovered from each fraction and hybridized to [“H]poly(U). (A) Particles prepared in 0.5 M KCl, released by puromycin; (B) particles prepared in 0.35 M KCl, released by 30 mM EDTA; (C) particles prepared as in (A) and subsequently treated with 30 mM EDTA.

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FRACTION NUMBER FIG. 6. Release of [‘HI-labeled RNA from polysomes by EDTA. Polysomes labeled with [ ‘Hluridine were pelleted and resuspended in TKM, and mRNA released by bringing the dissolved polysomes to 30 n&f EDTA as described under Materials and Methods. Release material was layered on 5-207 (w/v) linear sucrose gradients (in TKM) and centrifuged in an SW 41 rotor 40,000 rpm for 5 hr at 4°C. Aliquots of each fraction were precipitated with TCA and counted. p, AM; 0, [‘HI cpm.

units as well as in that of RNPs with varying salt concentrations, we always detected two peaks of radioactivity. Similarly, release with puromycin in 0.5 A4 KC1 yielded two peaks of labeled material with modal sedimentation values of 30-40 and 15 S. The protein content of [3H]-labeled particles released from polysomes was determined by isopycnic banding on CsSO4 gradients. Following fixation with formaldehyde material from the 30 and 15 S regions of sucrose gradients was centrifuged on preformed Cs$S04 gradients. It is important to note that even on preformed gradients 9 S histone mRNA requires 40-48 hr of centrifugation to reach equilibrium, twice the time required for the 21 S RNA. A typical result is shown in Fig. 7. Panels A and B represent the density of particles sedimenting at lo20 S which were released from polysomes by EDTA in 0.5 and 0.25 M KCl. Panel C shows the 15 S particles released from polysomes by puromycin in 0.5 M KCl. In all cases the density of the majority of the

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fixed material is 1.60-1.66 g/cm3, close to the density of extracted RNA (1.66 g/cm3). The minor peaks at density 1.45 g/cm3 represent labeled tRNA which, even after 2 days of centrifugation, does not band at a density expected for free RNA. We have done the same experiments with the 30 S fraction from sucrose gradients which also exhibits density of 1.62-1.66 g/ cm” (Fig. 8). Panels A and B present the density of particles sedimenting at 30 S which were released from polysomes by EDTA in 0.5 and 0.25 M KCI. Panel C presents the density of 30 S particles released from polysomes by puromycin in 0.5 M KCl. The majority of released material containing newly transcribed mRNA has a density of 1.62-1.67 g/cm”, again as is the case for 15 S particles, approaching the density of deproteinized RNA. The peak with a density of 1.45-1.50 g/cm3 present in the material released by puromycin (Fig. SC) is due to incomplete release and represents residual polysomes. Figures 7 and 8

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should be directly compared with Fig. 5 which presents the densities of particles containing oogenetic poly(A)+mRNA released from polysomes with puromycin and EDTA. In all cases most of the material containing newly transcribed RNA released by either EDTA or puromycin has a density approaching that of pure RNA. To show that our fixation procedures were appropriate, we fixed intact polysomes with formaldehyde. The labeled RNA banded at 1.44 g/cm”, as expected for mRNA bound to ribosomes (DeFilippes, 1965; Wilt et al., 1973). Determination of RNA Size in Material Released from Polysomes We have determined the size of RNA present in the various sedimentation classes of structures containing newly synthesized mRNA released from polysomes with EDTA. Gradients were divided into three fractions: (a) greater than 40 S, (b) 25-35 S, 17 1.6 1.5 1.4 13 12 II

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7. Buoyant density of fractions containing newly transcribed RNA sedimenting at 15 S following release from polysomes by EDTA or puromycin. Polysomes were isolated from embryos labeled with [“Hluridine as described under Materials and Methods except that the buffer contained 0.25 instead of 0.35 M KCI. Pelleted polysomes were dissolved in either 0.25 or 0.5 M KCl, both containing 5 mM Mg-acetate, 0.05 M triethanolamine, pH 7.6. Polysomes resuspended in 0.25 M KC1 were made 30 n&f EDTA while polysomes dissolved in 0.5 M KC1 were divided, with half brought to 30 mJ4 EDTA and half brought to 2 mJ4 puromycin, 0.1’S Triton X-100. Released material was centrifuged on 5-20s (w/v) linear sucrose gradients in the respective buffers and fractions sedimenting at 15 S from each gradient were fixed with formaldehyde and centrifuged on preformed Cs?SOI gradients for 40 hr. (A) Particles prepared in 0.5 M KCl, released with EDTA; (B) particles prepared in 0.25 M KCl, released with EDTA; (C) particles prepared in 0.5 M KCl, released with puromycin. FIG.

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FIG. 8. Buoyant density of fractions sedimenting at 30 S following EDTA or puromycin release of structures containing newly transcribed RNA. Polysomes were isolated after labeling with [‘Hluridine, as described under Materials and Methods except that the buffer contained 0.25 instead of 0.35 M KCl. Pelleted polysomes were dissolved in either 0.25 or 0.5 M KCI, both containing 5 mM Mg-acetate, 0.05 M triethanolamine, pH 7.6. Polysomes resuspended in 0.25 M KC1 were made 30 m&f EDTA while polysomes dissolved in 0.5 M KC1 were divided, with half brought to 30 mkf EDTA and half brought to 2 mM puromycin, 0.1% Triton X-100. Released material was centrifuged on 5-20% (w/v) linear sucrose gradients in the respective buffers and fractions sedimenting at 30 S were fixed with formaldehyde and centrifuged on preformed C&SO4 gradients for 40 hr. (A) Particles prepared in 0.5 M KCl, released with EDTA; (B) particles prepared in 0.25 M KCl, released with EDTA, (C) particles prepared in 0.5 M KCl, released with puromycin.

and (c) lo-20 S. Marker E. coli rRNA was added and the labeled RNA extracted as described under Materials and Methods. RNA was heated to 60°C for 10 min and sedimented on sucrose gradients in SDS buffer to minimize aggregation. The results (Fig. 9) show a direct relationship between sedimentation velocity of [3H]-labeled mRNA-containing structures and size of the corresponding RNA. Fractions with S values greater than 40 S contained RNA sedimentation with a peak at 21 S; 25-35 S regions contained the same high molecular weight RNA (21 S) as well as 9 S histone mRNA. That the 9 S RNA was indeed histone mRNA was confirmed by our observation (data not shown) of hybridization of this RNA with a labeled histone DNA probe prepared from the plasmid pSpl7 containing the genes coding for histones HzA and H3 (Kedes et al., 1975). The slowsedimenting fractions of gradients contained almost exclusively 9 S RNA. The structures containing high molecular

weight newly transcribed mRNA when released from polysomes sedimented in a broad peak centered at 30 S while the poly(A)+mRNA-containing mRNPs isolated under similar conditions from polysomes of actinomycin-D-treated embryos sedimented in a broad peak centered at 55 S. This difference in sedimentation was not due to differences in mRNA size since poly(A)+mRNA extracted from the 55 S particle fraction and centrifuged on SDS sucrose gradients had a modal S value of 21 S as previously reported for poly(A)‘mRNA extracted from the mRNPs of unfertilized eggs (Kaumeyer et al., 1978). Control for Direct Effect of Actinomycin on mRNP Sedimentation

D

An objection might be raised that actinomycin D used to suppress RNA synthesis caused the artifactual formation of the 55 S RNPs which contain the 21 S oogenetic poly(A)+mRNA released from polysomes in actinomycin-treated embryos. While

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Sea Urchin Embryo mRNPs

I 2

3 4

5 6 7 8 9 I

2 3 4

5 6 7

8

9

IO

FRACTION NUMBER FIG. 9. Sedimentation of newly transcribed RNA extracted from structures released from polysomes with EDTA. Sucrose gradients of released structures were divided into three sedimentation classes: (A) greater than 40 S, (B) 25-35 S, and (C) IO-20 S. E. coli rRNA was added and the labeled RNA was extracted as described. The RNA was dissolved in SDS buffer, heated at 60°C for 10 min and layered on 15-30s (w/v) linear sucrose gradients made in SDS buffer. Centrifugation was in an SW41 rotor at 40,000 rpm for 6.5 hr at 23°C. Gradients were fractionated, bovine serum albumin was added as carrier, and RNA was precipitated with TCA. -, Azw; 0, [3H]uridine-labeled RNA.

such an artifact appeared unlikely because of the lack of observable effect of actinomycin on protein synthesis in sea urchin embryos (Sargent and Raff, 1976), we devised a direct test to eliminate this possibility. The feasibility of directly comparing oogenetic poly(A)+mRNA with newly transcribed RNA in polysomes of embryos not exposed to actinomycin D is based on our calculations that the bulk of the poly(A)+mRNA on polysomes at 128cell stage is of oogenetic origin. Based on available data (see Davidson, 1976) the rate of heterogeneous nuclear RNA (HnRNA) synthesis by a Strongylocentrotus purpuratus embryo at 128-cell stage is approximately 1 and the fraction of pg/min/embryo HnRNA that is messageis 5%. The interval between the time when newly transcribed mRNA begins appearing on polysomes (Wilt, 1970), and the time we harvest the embryo is 5 hr, or enough time for the synthesis of 300 pg of HnRNA or 15 pg of mRNA. We have passed newly transcribed mRNA over an oligo(dT)-cellulose column and determined that only 10% of the RNA

transcribed at this stage is poly(A)+mRNA (data not shown). This is in close agreement with the results of Nemer (1975). The total amount of newly transcribed mRNA that is poly(A)+ is therefore 10% of 15 or 1.5 pg. There is approximately 70 pg of mRNA on polysomes (Nemer et al., 1974; Galau et al., 1977) of which 45% (31.5 pg) is poly(A)+mRNA. Thus only 5% (1.5/31.5 pg) of the poly(A)+RNA on polysomes represents newly synthesized poly(A)‘mRNA and 95% (30/31.5 pg) represents oogenetic mRNA. Polysomes of embryos labeled with [3H]uridine were isolated and treated with EDTA as described under Materials and Methods. Released mRNA-containing structures were centrifuged on sucrose gradients. The gradients were fractionated and aliquots of each fraction were precipitated with TCA to determine the distribution of labeled RNA. The remaining portions of each fraction were precipitated with ethanol and digested with proteinase K. Following precipitation and resuspension, each fraction was divided in two, with one set being hybridized to [3H]poly(U). Both

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DEVELOPMENTALBIOLOGY

sets were then digested with RNase A, precipitated with TCA, and counted. The results are shown in Fig. 10. The oogenetic mRNP fractions to which [3H]poly(U) hybridized show the distribution of poly(A)+mRNA-containing particles across the gradient, while unhybridized fractions indicate the negligible background of [“HI uridine-labeled RNA that is RNase A resistant. Also shown in Fig. 10, structures containing newly transcribed RNA have peaks at 25-35 and lo-20 S while oogenetic poly(A)+mRNPs sediment in a broad peak extending from 20 to 60 S with a peak center at 55 S. The distribution of poly(A)‘mRNPs exhibited in Fig. 10 is very similar to the distribution of oogenetic poly(A)+mRNPs released from the polysomes of embryos cultured in the presence of actinomycin D (Fig. 4). Since both newly transcribed mRNA of the 25-35 S particles and the oogenetic mRNA of the 20-60 S particles have the same sedimentation value (21 S) the difference in sedimentation of oogenetic and newly transcribed mRNAcontaining structures released from poly-

I

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somes is not due to culturing in actinomycin D or other artifacts of handling. Rather, the bulk of the poly(A)+mRNA in polysomes, while the same size as the newly synthesized RNA, is contained upon release from polysomes, in mRNP structures which differ in sedimentation characteristics and density from the mRNPs containing newly synthesized mRNAs released under the same conditions. DISCUSSION

Our results show a clear difference in association with proteins between oogenetic and newly transcribed mRNA. This difference is reconcilable with the dissimilar patterns of recruitment of these mRNAs. We have previously reported (Kaumeyer et al., 1978) that the poly(A)‘RNA of the unfertilized egg is contained in an RNP structure. The rise in protein synthesis following fertilization is due primarily to an increased availability of oogenetic mRNA (Humphreys, 1969, 1971; Brandis and Raff, 1978, 1979; Raff, 1979). It was proposed (Spirin, 1966) that changes in RNP struc-

345678910

FRACTION NUMBER FIG. 10. Sedimentation of structures containing poly(A)‘mRNA and newly transcribed RNA released from polysomes by EDTA. Polysomes labeled with [3H]uridine were pelleted, resuspended in TKM, and brought to 30 n-&f EDTA. Released material was centrifuged on 5-20s (w/v) linear sucrose gradients in an SW 41 rotor, 40,000 rpm for 5 hr, and fractionated. Aliquots of each fraction were precipitated with TCA to determine the distribution of newly transcribed RNA. The remainder of each fraction was digested with proteinase K, precipitated with ethanol, and resuspended in 2~ SSC. Each fraction was divided, with half being hybridized to [3H]poly(U). Both sets were digested with RNase A, precipitated with TCA, filtered, and counted. X, newly transcribed RNA, 0, poly(A)+mRNA; 0, [3H]uridine incorporated into RNase-A-resistant material.

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Sea Urchin Embryo mRNPs

ture (unmasking) regulated initiation of translation. We have utilized an in vitro protein synthesizing system derived from wheat germ to demonstrate the reality of masked forms of mRNA in the egg (Jenkins et al., 1978). Unmasking is an as yet undefined process which might be proposed to result in the partial deproteinization of mRNPs, or in modification or substitution of the proteins of the particles. If unmasking indeed controls the recruitment of mRNA into polysomes, the nonpolysomal fraction of oogenetic mRNA should remain in nontranslatable mRNP structures. We found that oogenetic poly(A)‘mRNA existing free in the embryo cytoplasm is contained in an mRNP with similar sedimentation and density characteristics as those found for particles extracted from unfertilized eggs by Kaumeyer et al. (1978). It is not certain, however, that all the poly(A)‘RNA of the cytoplasmic pool is destined to be translated since the complexity of the RNA in this fraction is greater than that of polysomal mRNA during early development (Hough-Evans et al., 1977). Messenger RNPs containing oogenetic poly(A)‘mRNAs were also isolated from polysomes. Particles isolated using puromycin plus 0.5 M K’ were found to contain more protein than particles released from polysomes with EDTA. Such a result is not surprising in view of the ability of EDTA to remove proteins from RNP structures (Spitnik-Elson and Atsmon, 1969; Kumar and Lindberg, 1972). Significantly, particles released by both methods contain protein, with the density of the puromycin-released particles (1.46 g/ cm”) being identical to that found by Kaumeyer et al. (1978) for mRNPs isolated from the cytoplasm of unfertilized eggs. Thus, the unmasking process does not result from a simple stripping of proteins from the mRNPs of eggs. There are a few reports of comparisons of the proteins of subpolysomal mRNPs

37

with those of mRNPs released from polysomes. Gander et al. (1973) compared RNPs containing globin mRNA from a subpolysomal homogenate with those released from polysomes and reported different proteins associated with the respective RNPs. Van Venrooij et al. (1977) reported that while the polysomal poly(A)+mRNAs of Ehrlich ascites tumor cells and rabbit reticulocytes have a 76,000-MW protein bound to their poly(A) segments, free cytoplasmic RNPs do not. Similarly, Vincent et al. (1977) found that duck globin mRNPs isolated from polysomes contain different proteins, including the poly(A)-binding proteins, than are found in free cytoplasmic globin mRNPs. Mazur and Schweiger (1978) have found a soluble protein in rat liver cells that has a high affinity for poly(A), and appears to be identical to the poly(A)-binding protein isolated from mRNPs. These reports suggest that unmasking might result in an exchange of proteins attached to oogenetic mRNAs. In contrast to oogenetic mRNAs, we found that newly synthesized mRNAs were associated with very little protein (at most 10% of particle mass). We cannot rule out the possibility that there are loosely bound proteins attached to the newly transcribed RNA and that these molecules become detached during the isolation procedure. In addition we cannot judge the effect of heparin as a possible competitor for RNP proteins, since the isolation of intact RNA required the presence of potent RNase inhibitor. Bentonite was found to drastically reduce the yield of poly(A)+RNA (Kaumeyer and Raff, unpublished observation). However, under conditions identical to those which allow us to isolate oogenetic RNA as protein-rich RNP, newly synthesized RNA appears to be stably associated with only a small amount of protein. Infante and Nemer (1968) in their study of sea urchin embryo subpolysomal mRNPs containing newly synthesized mRNAs reported densities in CsCl ranging from 1.5 to

38

DEVELOPMENTALBIOLOGY

1.8 g/cm3, suggesting a wide range in the amount of protein associated with RNA (from 0 to 50% protein with a peak center at about 1.62 g/cm3, corresponding to about 40% protein). Infante and Nemer (1968) demonstrated the association of protein with newly synthesized mRNAs by binding to nitrocellulose membrane filters and by showing an increase in particle density after digestion with Pronase. Our finding of a lower average protein content of particles containing newly synthesized mRNAs may arise from our use of longer centrifugation times (>48 hr) than those used by Infante and Nemer (lo-15 hr). When we used a centrifugation time of 16 hr, we found that particles containing mRNAs sedimenting at a mode of 21 S banded at a density of 1.60-1.64 g/cm3. However, particles containing 9 S mRNA had only reached a density of 1.45 g/cm3. Upon longer centrifugation these particles also attained equilibrium at the same density as the larger particles. The same effect has been noted by Greenberg (1977) with histone mRNPs prepared from L-cells. We did not assay the effect of proteases on the structures containing newly synthesized mRNAs because in our hands these structures already had a density approaching that of deproteinized RNA. Protease digestion did, however, cause oogenetic mRNPs to shift to high densities (Kaumeyer and Raff, unpublished data). We wish to stress again that we cannot rule out the possibility that there are loosely bound proteins attached to the newly synthesized RNA which become detached during the isolation procedure. However, these complexes must be weaker or qualitatively different from RNA-prooogenetic tein complexes containing mRNA, since under conditions which allow us to isolate oogenetic mRNA as stable protein-rich mRNPs newly transcribed mRNA is found in particles which contain a maximum of 10% protein. The possible signficance of oogenetic

VOLUME 72, 1979

mRNAs being contained in stable mRNPs qualitatively different from particles containing newly transcribed mRNAs is not that very different control mechanisms may govern the entry of these two classes of mRNA into polysomes. Unlike oogenetic mRNA, newly synthesized mRNA appears to enter polysomes rapidly once it reaches the cytoplasm. If newly synthesized mRNAs enter polysomes with no appreciable lag, the need to package this mRNA into a stable RNP particle as a means of storage and control is alleviated. This hypothesis is consistent with the finding of Hogan and Gross (1971) that protein synthesis is not required for newly synthesized mRNAs to enter polysomes in cleavage stage embryos. Dworkin and Infante (1976) and Dworkin et al. (1977) have suggested, on the basis of the kinetics of entry of newly synthesized RNAs into the free pool and into polysomes, that the RNA in the subpolysomal fractions does not serve as precursor to message. Dworkin and Infante (1976) used [“Hladenosine to label newly synthesized RNAs. Since the kinetics of poly(A) turnover are very different from those of mRNAs (Wilt, 1977; Dolecki et al., 1977), the exact relationship of newly synthesized subpolysomal RNA to polysomal RNA is still uncertain. The rapid utilization of newly transcribed RNA is in sharp contrast to utilization of oogenetic mRNAs where the mRNAs are present in the cytoplasm long before they are actually translated (Raff, 1979). It is in the latter that mRNPs would be expected to play an important regulatory role. We wish to acknowledge the competent and intelligent technical assistance of Mrs. Carolyn Huffman. We would also like to thank Dr. E. C. Raff for helpful and critical reviews of this manuscript. This work was supported by USPHS Grant HD 06902. R.A.R. is the recipient of USPHS Career Development Award K04 HD47. E.Y. was a Predoctoral trainee in Molecular and Cellular Biology supported by USPHS Award T32 GM 07227.

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cleoproteins from buoyant density measurements. In “Methods in Enzymology” (K. Moldave and L. Grossman, eds.), Vol. 20, pp. 512-521. Academic Press, New York. HOGAN, B., and GROSS, P. R. (1971). The effect of protein synthesis inhibition on the entry of messenger RNA into the cytoplasm of sea urchin embryos. J. Cell Biol. 49, 692-701. HOUGH-EVANS, B. R., WOLD, B. J., ERNST, S. G., BRITTEN, R. J., and DAVIDSON, E. H. (1977). Appearance and persistance of maternal RNA sequences in sea urchin development. Develop. Biol. 60, 258-277. HUMPHREYS, T. (1969). Efficiency of translation of messenger RNA before and after fertilization in sea urchins. Develop. Biol. 20,435-453. HUMPHREYS, T. (1971). Measurements of messenger RNA entering polysomes upon fertilization of sea urchin eggs. Develop. Biol. 26, 201-208. INFANTE, A. A., and KRAUSS, M. (1971). Dissociation of ribosomes induced by centrifugation: Evidence for doubting conformational changes in ribosomes. Biochem. Biophys. Acta 246,81-99. INFANTE, A. A., and NEMER, M. (1968). Heterogeneous ribonucleoprotein particles in the cytoplasm of sea urchin embryos. J. Mol. Biol. 32,543-565. JENKINS, N. A., KAUMEYER, J. F., YOUNG, E. M., and RAFF, R. A. (1978). A test for masked message: The template activity of messenger ribonucleoprotein particles isolated from sea urchin eggs. Develop. Biol. 63, 279-298. KAUMEYER, J. F., JENKINS, N. A., and RAFF, R. A. (1978). Messenger ribonucleoprotein particles in unfertilized sea urchin eggs. Develop. Biol. 63, 266278. KEDES, L. H., CHANG, A. C. Y., HOUSMAN, D., and COHEN, S. N. (1975). Isolation of histone genes from unfractionated sea urchin DNA by subculture cloning in E. coli. Nature (London) 255, 533-538. KUMAR, A., and LINDBERG, U. (1972). Characterization of messenger ribonucleoprotein and messenger RNA from KB cells. Proc. Nat. Acad. Sci. USA 69, 681-685. MAZUR, G., and SCHWEIGER, A. (1978). Identical properties of an mRNA-bound protein and a cytosol protein with high affinity for polyadenylate. Biothem. Biophys. Res. Commun. 80,39-45. NEMER, M. (1975). Developmental changes in synthesis of sea urchin embryo messenger RNA containing and lacking polyadenylic acid. Cell 6, 559-570. NEMER, M., GRAHAM, M., and DUBROFF, L. M. (1974). Co-existence of non-histone messenger RNA species lacking and containing polyadenylic acid in sea urchin embryos. J. Mol. Biol. 89, 435-454. PINDER, J. C., STAYNOV, D. Z., and GRATZER, W. B. (1974). Electrophoresis of RNA in formamide. Biochemistry 13, 5353-5378. RAFF, R. A. (1979). Masked messenger RNA and the

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regulation of protein synthesis in eggs and embryos. In “Cell Biology: A Comprehensive Treatise” (L. Goldstein and D. M. Prescott, eds.), Vol. IV. Academic Press, New York, in press. SARGENT, T., and RAFF, R. A. (1976). Protein synthesis and messenger RNA stability in activated, enucleated sea urchin eggs are not affected by actinomycin D. Develop. Biol. 48, 327-335. SHEPARD, H. M. (1977). “Oogenetic Histone Messenger Ribonucleic Acid: Its Form of Storage in Unfertilized Eggs of the Sea Urchin, Strongylocentrotus purpuratus.” Doctoral dissertation, Indiana University. SPIRIN, A. S. (1966). On “masked” forms of messenger RNA in early embryogenesis and in other differentiating systems. Curr. Top. Deoelop. Biol. 1, l-38. SPITNIK-ELSON, P., and ATSMON, A. (1969). Detachment of ribosomal proteins by salt. I. Effect of conditions on the amount of protein detached. J. Mol. Biol. 45, 113-124.

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VAN VENROOIJ, W. J., VAN EEKELEN, C. A. G., JANSEN, R. T. P., and PRINCEN, J. M. G. (1977). Specific poly(A)-binding protein of 76,000 molecular weight in polyribosomes is not present on poly(A) of free cytoplasmic mRNP. Nature (London) 270,189-191. VINCENT, A., CIVELLI, O., BURI, J.F., and SCHERRER, K. (1977). Correlation of specific coding sequences with specific proteins associated in untranslated cytoplasmic messenger ribonucleoprotein complexes of duck erythroblasts. FEBS Lett. 77, 281-286. WILT, F. H. (1970). The acceleration of ribonucleic acid synthesis in cleaving sea urchin embryos. Deuelop. Biol. 23, 444-455. WILT, F. H. (1977). The dynamics of maternal poly(A) containing mRNA in fertilized sea urchin eggs. Cell 11,673-681. WILT, F. H., ANDERSON, M., and EKENBERG, E. (1973). Centrifugation of nuclear ribonucleoprotein particles of sea urchin embryos in cesium sulfate. Biochemistry 12, 959-966.