Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

DEVELOPMENTAL

Cell-Free

BIOLOGY

60, 48-68 (1977)

Translation

Analysis Amphibian

of Messenger RNA in Echinoderm Early Development’

and

JOAN V. RUDERMAN~ AND MARY Lou PARDUE Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received December 28,1976; accepted in revised form May 19,1977 A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(Al+ mRNA, poly(A)- histone mRNA, and poly(A)- nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)+ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)- nonhistone mRNAs than do embryos. Some differences in the proteins encoded by poly(A)+ and poly(A)- RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)+ and poly(A)- RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous corn&ration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)mRNA, encoding a 70,000-80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)+ nonhistone mRNA, poly(A)- histone mRNA, and poly(A)+ histone mRNA. Amphibian ovary RNA apparently lacks an abundant poly(A)- nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding hi&me-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay. INTRODUCTION

a substantial acceleration in the amounts of both protein and RNA synthesis (Epel, The mature sea urchin egg contains a 1967; Humphreys, 1969, 1971). During the store of “maternal” mRNA which is prefirst few cleavage divisions, a major part of vented, in some as yet unknown manner, protein synthesis is directed by maternal from directing protein synthesis prior to mRNA which is recruited from the subrifertilization (reviewed by Gross, 1967). bosomal fraction of the embryo (HumMost, if not all, of the stored maternal phreys, 1971). As development proceeds, mRNA, which includes histone mRNA and polyadenylated RNA, is localized in newly transcribed mRNA makes an inthe subribosomal fraction of the egg (Sla- creasing contribution to embryonic protein ter et al., 1973a, b; Wilt, 1973; Gross et al ., synthesis. Histone mRNA makes up a sub1973a, b; Fyrberg and Ruderman, 1976). stantial fraction of the mRNA synthesized Fertilization of the sea urchin egg triggers by rapidly cleaving embryos. Both qualitative and quantitative changes in the syn1 An abstract of this work has been previously thesis of histone mRNA, poly(A)- nonhispublished (J. Cell Biol. 70, 8a, 1976). 2 To whom correspondence should be addressed at tone m.RNA, and poly(A)+ mRNA occur during early development (Kedes and her present address: Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115. Gross, 1969b; Nemer and Lindsay, 1969; 48 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0012-1606

RUDERMAN AND PARDUE

Gross et al., 1973a; Ruderman et al., 1974; Nemer et al., 1974; Nemer, 1975). Hybridization studies by Galau et al. (1976) show that some changes occur in the sets of structural genes expressed at different stages of early sea urchin embryonic development, although some genes are active throughout early development. Their studies, however, examine primarily the “rare, complex” mRNA sequence class composed of a very large number of different kinds of mRNA sequences, each of which is present in very few copies per cell. The cell-free translation assay, on the other hand, almost certainly analyzes the more “abundant” mRNA sequences. The translation assay has previously been used to demonstrate developmental changes in the mRNA encoding the lysine-rich histone class, Hl (Ruderman et al., 1974; Arceci et al., 1976). In this report, we have used the translation assay to monitor changes in the abundant poly(A)+ and poly(A)- mRNA classes during early sea urchin embryonic development. The mature amphibian oocyte also contains a store of mRNA which is synthesized and accumulated during oogenesis (Davidson, 1968; Rosbash and Ford, 1974; Somerville and Malcolm, 1976). Most of the polyadenylated mRNA is stored in the subribosomal supernatant of the oocyte (Rosbash and Ford, 1974; Darnbrough and Ford, 1976). Xenopus oocytes synthesize and accumulate a pool of histone proteins during oogenesis which are used during later embryonic development (Adamson and Woodland, 1974). Following maturation and fertilization, the rate of protein synthesis rises only gradually, and no striking changes in mRNA transcription have been detected. In particular, no transcription of histone mRNA during the rapid cleavage stage of embryogenesis has been observed (Davidson, 1968; Brown and Littna, 1964, 1966; Bacharova et al., 1966; Gurdon and Woodland, 1969; Woodland, 1974). Thus, in contrast to sea urchins (Kedes and Gross, 1969b; Benttinen and

mRNA

in Early

Development

49

Comb, 1971: Skoultchi and Gross, 1973; Cognetti et al., 1974)) the bulk of the histone complement which associates with the embryonic nuclei of amphibians is recruited from a stored histone protein pool of the oocyte and from histones synthesized on maternal histone mRNA after maturation and fertilization. We have used the cell-free translation assay to compare the spectra of abundant translatable mRNA sequences in poly(A)+ and poly(A)- RNAs of amphibian ovaries and oocytes. We have also examined the spectra of the more abundant mRNA sequences present at different stages of amphibian oogenesis and have determined the stage at which mRNA encoding histone-like proteins is first detectable. METHODS

Animals. Lytechinus pictus were purchased from Pacific Biomarine, Venice, California. Arbuciu punctulutu were obtained from Woods Hole, Massachusetts. Triturus viridescens females were purchased from Lee’s Newt Farm, Oak Ridge, Tennessee. Xenopus luevis females were obtained from our laboratory stock. Collection of sea urchin gametes, fertilization, and embryo culture were performed as described by Kedes and Gross (1969a). Postfertilization polyadenylation was inhibited with 3’-deoxyadenosine (cordycepin), as described by Mescher and Humphreys (1974). Washed unfertilized eggs were cultured for 1 hr in sterile seawater containing 750 pg/ml of cordycepin. Eggs were then fertilized, and the embryos were cultured continuously in the same solution to the morula stage and were processed as described below. This high concentration of cordycepin is required to achieve >95% inhibition of polyadenylation (Mescher and Humphreys, 1974). Isolation of RNA. Sea urchin eggs and embryos were collected by centrifugation, washed twice with calcium- and magnesium-free seawater and once with 0.2 M NaCl, 5 mM Mg(OAc),, 20 mM Tris, pH

50

DEVELOPMENTAL BIOLOGY

7.6 (TNM), and homogenized in 10 vol of TNM + 0.25% diethylpyrocarbonate (DEP). The homogenate was centrifuged at 12,000g for 15 min, and the supernatant was precipitated with 2.5 vol of EtOH at -20°C. The precipitate was dissolved in SDS buffer (0.5% SDS, 2 mM EDTA, 0.1 M NaCl, 20 mM Tris, pH 7.6). RNA was extracted with phenol and 4% isoamyl alcohol-chloroform, as described by Hogan and Gross (1971). Xenopus ovaries were removed and washed twice in Barth X solution (Barth and Barth, 1959), and oocytes were obtained as described by Rosbash and Ford (1974). Oocytes were manually isolated and staged according to Dumont (1972). Oocytes or ovary fragments were washed in 0.3 M NaCl, 10 mM MgC12, 20 mM Tris, pH 7.4, and were homogenized in 10 vol of the same buffer containing 0.25% DEP. The homogenate was centrifuged at 300g for 15 min, and the supernatant was precipitated with EtOH and was resuspended in SDS buffer; RNA was extracted. Triturus ovaries were removed, washed twice with TBS (75 mM KCl, 25 n&f NaCl, 10 n-&f Tris, pH 7.4), and homogenized in 10 vol of TBS containing 0.25% DEP. RNA was isolated from the 300g supernatant as above. All RNA preparations had A26,,/A28,, ratios >2.0. Purified sea urchin embryonic polysomal histone mRNA, labeled with [3H]uridine, was prepared as described by Gross et al. (1973a). Okgo(cellulose fractionation of RNA. RNA was fractionated into poly(A)+

and poly(A)- RNA classes by oligo(dT)cellulose (Collaborative Research, Waltham, Massachusetts) column chromatography as described by Aviv and Leder (1972) and Singer and Penman (1973). All fractionations were done using the same batch of oligo(dT)-cellulose. The poly(A)+ RNA fractions were precipitated in the presence of 40 pglml of purified yeast tRNA. All fractionations were monitored by hybridization with [3Hlpoly(U) (gift of R. C. Herman), as described by Milcarek et

VOLUME 60, 1977

al. (1974), and were found to be >95% com-

plete. In vitro translation. RNA preparations were suspended in 0.2 M NaCl and were precipitated with EtOH. The precipitates were washed three times with 80% EtOH at -20°C and once with 100% EtOH and were lyophilized. The RNA was suspended in glass-distilled water to final concentrations of 3 mglml for unfractionated RNA or poly(A)- RNA or lo-100 pg/ml for poly(A)+ RNA, except in the instances when Xenopus oocyte RNA was used. RNA preparations were stored at -70°C. RNA was translated in a wheat germ cellfree protein-synthesizing system as described by Roberts and Paterson (1973), modified by the addition of spermine (Sigma) to a final concentration of 40 j&f. The presence of polyamines improves the in vitro translation of higher molecular weight RNAs (Atkins et al., 1975; Roberts et al., 1975). All reactions were carried out using aliquots of the same preparation of wheat germ S-30. Ten-microliter reaction mixtures contained 6 Fg of unfractionated or poly(A)- RNA or lo-100 ng of poly(A)+ RNA, as indicated in the relevant figure legends, and either 4 PCi of [35Slmethionine (Amersham, 280 Ci/ mmole) or 4 &i of i3Hllysine (New England Nuclear, 55 Ci/mmole). Reactions were incubated for 90 min at 20°C and were terminated by freezing at -70°C. One microliter of reaction mixture was assayed for 5% hot trichloroacetic acid-precipitable incorporation. Polyacrylamide gel electrophoresis . Aliquots of translation products were mixed with an equal volume of 1% SDS, 30% glycerol, 200 mM dithiothreitol, 0.01% bromphenol blue, 50 mM Tris, pH 6.8, and were heated for 60 set in a boiling water bath. Samples were analyzed on SDS 12.5% acrylamide slab gels (1.5 mm thick) as described by Laemmli (1970). Translation products were also examined by electrophoresis on 2.5 M urea-acetic acid (pH 2.7), acrylamide (15 or 18%) slab gels con-

RUDERMAN AND PARDUE

taining 0.16% ammonium persulfate (Panyim and Chalkley, 1969). Crystal violet was used as dye marker. Aliquots of translation products were usually mixed with an equal volume of 5 M urea, 1.8 N acetic acid, 5 n&f dithiothreitol at 4°C and were heated briefly at 37°C prior to electrophoresis. This method, which gives variable recovery of histone H4 and variable resolution of in vitro product histones H3, H2B, and H2A on the gels, was used to obtain the data presented in Fig. 8. In one case (Fig. 4), product was precipitated with 20% trichloroacetic acid, followed by solubilization of the acetone-washed precipitate in 5 M urea, 5 mit4 ammonium bicarbonate, 0.5% P-mercaptoethanol, 0.9 N acetic acid, 5% glycerol prior to electrophoresis. This method gives substantially better recovery and resolution of the histones on acid-urea gels (H. R. Woodland, personal communication). Gels were stained with 0.2% Coomassie brilliant blue R in 30% methanol, 10% acetic acid, and were destained. Calf thymus and sea urchin embryo histones were used as markers on both types of gels. While the electrophoretic mobilities of amphibian and calf histones are not completely identical, they are similar enough to be of use as markers (Destree et al., 1972, 1973; Adamson and Woodland, 1974; Destree, 1975). Electrophoresis of individual calf thymus histone subfractions, the gift of Fran Jurnak, showed that, on both types of gels, calf thymus histones migrate in the following order (lowest to highest electrophoretic mobility): Hl, H3, H2B, H2A, H4. Gels containing [YSlmethionine-labeled in uitro products were dried and exposed against RP Royal X-Omat X-ray film. Gels containing 13Hllysine-labeled product were autofluorographed using the method of Bonner and Laskey (1974) and were exposed against X-ray film which had been presensitized to linearity (Laskey and Mills, 1975). Liquid scintillation determination of radioactivity in polyacrylamide slab gels. In

mRNA

in Early

Development

51

some cases, the amount of incorporation of 13Hllysine into histone and nonhistone translation products encoded by unfractionated, poly(A)+ and poly(A)- RNAs was determined. The histone and nonhistone regions of individual lanes of dried slab gels were excised and cut into 3 x 3-mm pieces. Individual pieces were placed in scintillation vials and rehydrated with 1 ml of distilled water for 45 min. Excess water was removed, 0.5 ml of Protosol (New England Nuclear) was added, and capped vials were incubated at 45°C for 16 hr. Ten milliliters of toluene containing 4% Liquifluor (New England Nuclear) was then added, and samples were counted. RESULTS

OligotdTl-Cellulose Fractionation Poly(A) + and Poly(A) - RNA

of

RNA was isolated from the postmitochondrial(12,OOOg) supernatants of sea urchin eggs and embryos and from the 300g supernatants of Xenopus and Triturus ovaries and was fractionated into poly(A)+ and poly(A)- RNA classes by oligo(dT)cellulose column chromatography. Separation of poly(A)+ from poly(A)- RNA was judged to be reasonably complete. More than 95% of the poly(A)-containing RNA sequences applied to the column were recovered in the poly(A)+ RNA fraction in each experiment, as monitored by [3Hlpoly(U) hybridization. Contamination of poly(A)+ RNA by poly(A)- RNA was more difficult to rule out. As a test of the fractionation procedure, r3H]uridine-labeled purified sea urchin (Lytechinus pictus) embryonic polysomal poly(A)- histone mRNA was fractionated both alone and mixed with unlabeled postmitochondrial supernatant morula RNA prior to fractionation. In both cases, less than 3% of the radioactivity was recovered in the poly(A)+ RNA fraction. Therefore, at least this one class of poly(A)- mRNA does not bind to oligo(dT)-cellulose , either nonspecifically or via aggregation with poly(A)-+ RNA. Ribosomal RNA also provides a

52

DEVELOPMENTAL BIOL~CY

measure of efficiency of RNA fractionation on oligo(dT)-cellulose. A small amount (2-3%) of the ribosomal RNA eluted from the column with poly(A>+ RNA. RNA fractionations were checked by two other criteria. In one experiment, Triturus ovary RNA was divided into two equal parts. One part was chromatographed on oligo(dT)-cellulose under standard conditions. The second part was treated with 90% dimethylsulfoxide (DMSO), 10 mM Tris, pH 7.5, at 60°C for 5 min prior to oligo (dT)-cellulose fractionation in order to disrupt any aggregates of poly(A)- RNA with poly(A)+ RNA which might be present. The spectra of proteins encoded in vitro by the control poly(A)+ and poly(A)- RNAs and by the poly(A)+ and poly(A)- RNAs obtained after DMSO treatment were examined by autoradiography of one-dimensional SDS-polyacrylamide gels. The DMSO treatment of total ovarian RNA had no detectable effect on the subsequent distribution of template-active RNAs (including histone mRNAs) into the poly(A)+ and poly(A)- RNA fractions obtained by oligo(dT)-cellulose chromatography (data not shown). In another experiment using Xenopus ovary RNA, poly(A)+ and poly(A)- RNAs obtained by the standard fractionation procedure were each subjected to a second cycle of fractionation on oligo(dT)-cellulose. The resultant RNA fractions were assayed for template activity and for the spectra of 13Hllysine-labeled proteins encoded in vitro by SDS gel electrophoresis and fluorography. Examination of the fluorograms, both visually and by direct determination of the radioactivity in the histone and nonhistone regions (as described in Methods) revealed that virtually all the detectable template-active RNA sequences fractionating as poly(A)+ RNA in the first oligo(dT)-cellulose cycle remained in the poly(A)+ RNA fraction in the second cycle. Similarly, almost all of the sequences present in the first poly(A)V RNA fraction were recovered in the poly-

VOLUME 60, 1977

(A)- RNA fraction following the second oligo(dT)-cellulose binding and elution cycle. Cell-Free Translation of Sea Urchin and Amphibian Ovary RNA

Egg

RNA fractions were translated in a wheat germ cell-free protein-synthesizing system using either [3Hllysine or P5Slmethionine as tracer, and the incorporation of labeled amino acids into protein was measured (Table 1). Except when noted, lo-cl.1 reaction mixtures contained 600 pglml of unfractionated or poly(A)RNA or 1-5 pglml of poly(A)+ RNA. For each individual RNA fraction, the in vitro incorporation was roughly proportional to the amount of RNA added within the ranges of 100-800 pg/ml for unfractionated or poly(A)- RNA and l-10 pg/ml of poly(A)+ RNA (Fig. 1). Incorporation was linear for at least 90 min at 20°C. The cellfree translation assay is not, however, strictly quantitative in the amount of incorporation directed by mRNA (Table 1). The ratio of incorporation per microgram of added RNA varied with species and, in the case of amphibians, with different preparations. In almost all instances, the in vitro template activity of unfractionated RNA was less than the sum of the template activities of the separated poly(A)+ and poly(A)- RNA components (Table 1). This phenomenon may be due to general or specific effects (either stimulatory or inhibitory) of other RNA components on mRNA template activity (e.g., Jacobs-Lorena and Baglioni, 1972; Wettenhall and Slobbe, 1976; Zilberstein et al; 1976). Since we do not know the in vitro translational efficiencies of mRNA contained within the unfractionated, poly(A)+ and poly(A)preparations, we can make no firm quantitative conclusions about the amounts of particular mRNAs in various preparations. Unfractionated RNAs from sea urchin eggs and from Xenopus and Triturus ovaries direct the synthesis of a wide range of

RUDERMAN AND PARDUE

mRNA TABLE

in Early

53

Development

1

In vitro INCORPORATION OF [T~]METHIONINE AND [3H]L~~~~~ DIRECTED BY RNA FROM SEA URCHIN (Lytechinus pictus OR Arbacia punctulata) EGGS AND EMBRYOS, Xenopus laevis OVARY AND OOCYTES, AND Triturus viridescens OVARY~ A. Sea urchin egg and amphibian ovary RNA stage

Species

Lytechinus

Egg

Xenopus

Ovary

Triturus

Ovary

stage

Egg

Morula

Cordycepin-treated

Gastrula

Stage*

1 2 4 6 Whole ovary

RNA

Unfractionated Poly(A)+ Poly(A)Unfractionated Poly(A)+ Poly(A)F Unfractionated Poly(A)+ Poly(A)F No RNA added B. Arbacia egg and embryo RNA RNA

Unfractionated Poly(A)+ Poly(A)Unfractionated Poly(A)+ Poly(A)F morulae Unfractionated Poly(A)+ Poly(A)Unfractionated Poly(A)+ Poly(A)No RNA added C. Xenopus oocyte RNA RNA

Unfractionated Unfractionated Unfractionated Unfractionated Unfractionated No RNA added

lSHILysine incorporation kpm/l pl)

[VIIMethionine incorporation (cpm/l PI)

5,615 6,210 5,600 15,139 13,267 3,913 10,622 13,930 5,202 942

20,280 18,408 17,680 56,129 83,476 7,501 30,106 36,243 10,117 2,907

PHlLysine incorporation (cpm/l PI)

[‘SlMethionine incorporation kpmll cl11

6,745 10,388 5,065 24,485 14,780 21,310 8,900 8,115 8,480 22,410 28,538 19,549 590

24,108 35,000 15,920 40,700 50,342 23,792 15,300 22,326 18,500 47,500 91,000 31,132 3,011

13HlLysine incorporation (cpdl PII

Incorporation/ /a RNA (cpm)

5,333 16,204 9,800 18,218 22,129 1,907

13,203 3,108 3,038 3,975 4,040 -

a Postmitochondrial supernatant RNA from sea urchin eggs and embryos, and 300g supernatant RNA from Xenopus ovary and oocytes and Triturus ovary was prepared, fractionated by oligo(dT)-cellulose column chromatography (parts A and B), and translated in vitro as described in Methods. Translation reaction mixtures of 10 ~1 contained: (A) 6 pg of unfractionated or poly(A)- RNA or the amount of poly(A)+ mRNA obtained from 6 rg of unfractionated RNA, 12 ng of sea urchin egg poly(A)+ RNA, 53 ng ofxenopus ovary poly(A)+ RNA, and 18 ng of Triturus ovary poly(A)+ RNA; (B) 6 pg of unfractionated or poly(A)- RNA or the amount of poly(A)+ RNA (12-43 ng) obtained from 6 pg of unfractionated RNA from each stage; (C) unfractionated RNA from Stage 1 oocytes (0.26 pg), Stage 2 (4.6 pg), Stage 4 (2.6 pg), Stage 6 (4.1 pg), whole ovary (5 pg). One microliter of in vitro product was assayed for 5% TCA-precipitable incorporation. b Dumont (1972).

54

DEVELOPMENTAL BIOLOGY VOLUME60, 1977 20.

X--X O-0 O-O

0

unfrociionoted RNA poly(A)’ RNA p+(A)RNA

x

//

/A.“-./ fix,, I

//O

unfractionated

z polyCA)K RNA

“g poly F:,+

RNA

IO 100

I

FIG. 1. Cell-free incorporation of [?‘S]methionine directed by increasing amounts of Triturus unfractionated poly(A)+ and poly(A)- RNA. The amount of 5% trichloroacetic acid-precipitable incorporation in 1~1of translation product is plotted vs the amount of RNA added per 10 J of reaction mixture.

a

b

c

protein products in vitro (Figs. 2 and 3). Very little protein synthesis is observed in the absence of added RNA, although a small amount of endogenous incorporation into a 10,000 MW protein is observed following long exposure times. Both the poly(A)+ and poly(A)- fractions of sea urchin egg mRNA direct the cell-free synthesis of a variety of polypeptides. Sea urchin egg poly(A)- RNA promotes synthesis of material corn&rating with histones, as well as a wide range of higher molecular weight nonhistone proteins. Some differences in the spectra of proteins encoded by poly(A)+ and poly(A)- RNAs are apparent. Many of the products encoded by poly(A)+ and poly(A)- egg RNAs, however, exhibit similar electrophoretic mobilities when

defghij

FIG. 2. SDS gel electrophoresis of 5 ~1 of translation product labeled with [T3]methionine directed by Lytechinus egg RNA and by Xenopus and Triturus ovary RNA, as described in Table 1. Dots mark the positions of marker sea urchin histones; bars mark the positions of calf thymus histones, which migrate (top to bottom) Hl, H3, H2B, H2A, H4. Product encoded by: (a) Lytechinus egg unfractionated RNA, (b) Lytechinus egg poly(A)+ RNA, (c) Lytechinus egg poly(A)- RNA; (d) Xenopus ovary unfractionated RNA, (e) Xenopus ovary poly(A)+ RNA; (f) Xenopus ovary poly(A)- RNA, (g) Triturus ovary unfractionated RNA; (h) Triturus ovary poly(A)+ RNA, (i) Trirurus ovary poly(A)- RNA, (j) no RNA added. Exposures: (a-c) 6 days; (d-j) 3 days.

RUDERMAN

AND PARDUE

mRNA in Early Development

55

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DEVELOPMENTAL BIOLOGY

analyzed by one-dimensional gel electrophoresis. The apparent overlap in the RNA sequence content of the poly(A)+ and poly(A)- fractions of sea urchin eggs is too great to be attributed to incomplete fractionation of the RNA on oligo(dT)-cellulose. Furthermore, differences in the relative incorporation into the various protein bands apparently encoded in vitro by both egg poly(A)+ and poly(A)- RNAs, as well as patterns of incorporation directed by fractionated RNA preparations from other sources, argue against overlap due to poor separation of the two RNA classes. It is likely that some of the apparently similar product bands are due to fortuitous comigration of nonidentical proteins on onedimensional gels. The possibility exists, however, that sea urchin egg poly(A)+ and poly(A)- RNAs do, in fact, contain a number of mRNAs in common. This is in apparent contrast to the situation described for sea urchin embryonic polysomal RNA in which hybridization experiments show little or no overlap between blastula polysomal poly(A)+ and poly(A)RNAs (Nemer et al., 1974). Xenopus and Triturus unfractionated RNAs also direct incorporation into a wide range of protein products (Figs. 2-4). Examination of the cell-free translation products encoded by amphibian ovary poly(A)+ and poly(A)- RNAs shows that the majority of the detectable translation products are encoded by poly(A)+ RNA. The only discrete easily detectable products encoded by amphibian ovary poly(A)- RNA are those comigrating with marker histones; no more than slight and diffuse incorporation is observed in the nonhistone region of the gel. This is in contrast to the situation in the sea urchin egg in which poly(A)RNA directs the cell-free synthesis of a considerable number of discrete nonhistone proteins. We emphasize that the cellfree translation assay used here almost certainly would not detect mRNA sequences present in relatively low amounts.

VOLUME 60, 1977

HI

1

H3 H2B, H2A

a

b

FIG. 4. Acid-urea electrophoresis of gel [3H]lysine-labeled translation product (15 pl), encoded by Xenopus ovary poly(A)+ and poly(A)RNA, as described in Table 1. Translation product was precipitated with 20% trichloroacetic acid prior to electrophoresis on a 2.5 M urea, 0.9 N acetic acid, 18% polyacrylamide gel (see Methods). Bars indicate the mobility of Xenopus histones. Product encoded by: (a) Xenopus ovary poly(A)+ RNA; (b) Xenogus ovary poly(A)- RNA. Exposure: 15 days.

It is possible that amphibian ovary RNA contains a variety of rare nonhistone poly(A)- mRNAs. In order to determine more precisely the relative incorporation of 13Hllysine into histone and nonhistone proteins directed by poly(A)+ and poly(A)V RNAs, the histone and nonhistone regions of individual lanes of dried SDS-polyacrylamide slab gels were rehydrated, solubi-

RUDERMAN AND PARDUE

mRNA

lized, and counted (Table 2). Approximately 74% of the [3Hllysine-labeled products encoded by Xenopus ovary poly(A)RNA are found in the histone regions of the gel, whereas only 36% of the [3H]lysine-labeled products encoded by sea urchin (Arbacia punctulutu) egg poly(A)RNA corn&rate with histones. These data suggest that amphibian ovary RNA does not contain a component of abundant translatable nonhistone poly(A)- mRNAs as substantial as that found in the sea urchin egg. Both poly(A)+ and poly(A)- RNAs from amphibian ovary direct a considerable amount of incorporation into histone-like proteins (Figs. 2-4 and Table 2). It is likely that a large fraction of this product is, in fact, histone, as judged by several criteria. There is a substantial in vitro incorporation into material migrating like histones on both SDS and acid-urea gels (Figs. 24). As analyzed on SDS gels, the histonelike cell-free products exhibit a high lysine/methionine incorporation ratio, as compared to the higher molecular weight nonhistone cell-free products. When L3HlTABLE

in Early

57

Development

lysine-labeled translation product is examined by acid-urea gel electrophoresis, it is clear that both poly(A)+ and poly(A)RNAs stimulate incorporation into material which migrates much like Xenopus histones (Fig. 4). The slightly higher mobility of some of the hi&one-like translation products may be due to the lack of Nterminal acetyl groups (H. R. Woodland, personal communication). 13Hllysine-labeled product directed by Xenopus RNA contains material migrating as a single band in the Hl region on SDS gels (Fig. 3), but as a doublet in the Hl region on acidurea gels (Fig. 4), consistent with the electrophoretic mobility of Xenopus histone Hl (Adamson and Woodland, 1974; Destree, 1975; Jacob et al., 1976).Xenopus Hl lacks methionine (Destree, 1975). When cell-free product is labeled with [35S]methionine, no incorporation into the Hl region on either SDS (Fig. 2) or acidurea gels (data not shown) is observed. Furthermore, [3Hllysine-labeled material comigrating with marker histones shows acid solubilities characteristic of histones; the putative histone products are ex2

L3HlLys1~~ INCORPORATION INTO HISTONE AND NONHISTONE TRANSLATION PRODUCTS ENCODED BY Xenopus OVARY RNA AND BY SEA URCHIN EGG AND EMBRYO RNA” RNA translated

Xenopus ovary Unfractionated RNA Poly(A)+ RNA Poly(A)- RNA Arbacia egg Unfractionated RNA Poly(A)+ RNA Poly(A)- RNA Arbacia embryo (morula) Unfractionated RNA Poly(A)+ RNA Poly(A)F RNA

Total radioactivitv extracted f&m slab gel lane (cpm)

Radioactivity extracted from histone region (cpm)

Radioactivity extracted from nonhistone region (cpm)

Histone cpm/total cpm (%)

58,104 46,085 22,664

34,735 31,728 6,364

23,369 14,357 16,300

40 31 74

29,880 26,537 35,003

23,913 22,116 22,510

5,967 4,421 12,503

20 17 36

87,754 81,170 90,174

30,560 61,129 27,224

57,194 20,041 62,950

66 25 70

a Translation product labeled with [3H]lysine encoded by unfractionated, poly(A)+ and poly(A)- RNA from Xenopus ovary and from Arbacia eggs and morula-stage embryos was electrophoresed on SDSpolyacrylamide slab gels. After the gels had been used to obtain fluorographs, individual lanes of the dried gel were divided into histone and nonhistone regions and the radioactivity in each region was determined as described in Methods.

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DEVELOPMENTAL BIOLOGY

VOLUME 60, 1977

tracted with 0.4 N sulfuric acid, and product identified as Hl is selectively extracted with 5% perchloric acid (data not shown). Levenson and Marcu (1976) have recently used tryptic peptide analysis to characterize the proteins encoded by Xenopus ovary poly(A)+ RNA and conclude, in agreement with our findings, that histones are encoded by RNA found in both the poly(A)+ and poly(A)- RNA classes. Poly(A) + Histone

mRNA in Sea Urchins?

The demonstration of poly(AP histone mRNA in amphibian oocytes prompted a preliminary analysis of sea urchin poly(A)+ mRNA for its ability to direct histone synthesis in vitro. Lytechinus pictus morula polysomal RNA and its p01y(A)~ and poly(A)- components were translated in the wheat germ cell-free system. The 13H]lysine-labeled products of the a D c cell-free translation were analyzed by FIG. 5. SDS gel electrophoresis of F3Hllysine-laSDS-polyacrylamide gel electrophoresis (Fig. 5). While most of the in vitro histone beled translation of Lytechinus morula polysomal RNA. Dots mark the positions of marker sea urchin synthesis is directed by poly(A)V RNA, a histones. Product encoded by: (a) unfractionated rather substantial amount of Hl histone is RNA (75,000 cpm); (b) poly(A)+ RNA (125,000 cpm); encoded by RNA fractionating as poly(A)+ (c) poly(A)V RNA (90,000 cpm). Exposure: 8 days. RNA, as well as by poly(A)- RNA. Thus, in Lytechinus morulae, histone Hl ap- and poly(A)- RNA were analyzed by SDS pears to be encoded by both poly(A)+ and slab-gel electrophoresis (Table 1 and Fig. poly(A)- mRNAs, whilethe other four his- 6). Cell-free translation products directed tone classes are encoded predominantly by by egg, morula, and gastrula RNA show poly(A)- mRNA. In contrast to Lyte- distinct quantitative and, perhaps, qualitative differences, although most product chinus, poly(A)+ RNA from Arbacia punctulata directs [3H]lysine incorporation into bands seem to be encoded by RNAs present material corn&rating with all of the major at all three stages. Of particular interest is an egg poly(A)histone classes (Fig. 6). encoding a 70,000~80,000 MW protein of Cell-Free Translation Analysis of Changunknown function (“band 1,” Fig. 7). This ing Populations of mRNA in Sea Ur- mRNA is one of the more abundant chin Embryogenesis mRNAs in the egg, but is not detectable in morula and later-stage embryos, as judged We have used the cell-free translation assay to screen for changes in abundant by examination of the cell-free translation poly(A)+ and poly(A)- mRNAs during products (Figs. 6 and 7). More recent data early sea urchin development using the demonstrate that this particular mRNA is species Arbacia punctulata. The spectra of lost at some point between the 16- and 64[3H]lysine-labeled translation products en- cell stage (Fryberg and Ruderman, 1976). coded by Arbacia egg, morula, and gas- Cordycepin-mediated inhibition of postfercytoplasmic polyadenylation trula unfractionated RNA, poly(A)+ RNA, tilization

RUDERMAN AND PARDUE

mRNA

II r

in Early Development

. .. .

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DEVELOPMENTALBIOIJXY

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band I abcdefghij

k

I

m

FIG. 7. SDS gel electrophoresis of 5 ~1 of [Wlmethionine-labeled translation product of Arbaciu egg, morula, cordycepin-cultured morula, and gastrula stage embryo RNA, as described in Table 1. Only the top quarter of the gel is shown. Product encoded by: (a) egg unfractionamd RNA; (b) egg poly(A)+ RNA; (c) egg poly(A)- RNA, (d) morula unfractionated RNA; (e) morula poly(A)+ RNA; (f) morula poly(A)- RNA, (g) cordycepin-morula unfractionated RNA; (h) cordycepin-morula poly(A)+ RNA, (i) cordycepin-morula poloRNA; (j) gastrula unfractionated RNA; (k) gastrula poly(A)+ RNA; (1) gastrula poly(A)- RNA; (m) no RNA added. Exposure: 4 days.

does not prevent the disappearance of this mRNA activity from the poly(A)- RNA class during early cleavage, suggesting that this mRNA is not polyadenylated following fertilization (Fig. 7). In contrast to the range of products encoded by sea urchin egg poly(A)- RNA, morula and gastrula poly(A)- RNAs appear to encode fewer detectable nonhistone products in vitro (Fig. 6 and Table 2). No striking changes in the nonhistone products directed by poly(A)- RNA are observed between the morula and gastrula stages. Cell-Free Translation Analysis of mRNA Populations in Amphibian Oogenesis Xenopus oocytes free from adhering follicle cells were isolated and staged as described in Methods. RNA was extracted from each oocyte class and was translated in the cell-free system. The translation products labeled with 13H]lysine were analyzed on SDS and acid-urea gels, as shown in Figs. 8 and 9. There are no apparent striking qualitative or quantitative differences in the spectra of proteins encoded by RNA obtained from Xenopus oocytes of different stages when their cell-free products, labeled with 13Hllysine (or with [35S]methionine, data not shown) are examined by one-dimensional SDS-gel electrophoresis. This suggests that few stagespecific differences in the more abundant mRNA species occur during Xenopus oo-

genesis, in direct agreement with the data of Darnbrough and Ford (1976). It is, of course, possible and not unlikely that qualitative and quantitative changes in rarer mRNA sequences do occur during oogenesis. Such differences would not be detected by the translation assay. However, the data do demonstrate that changes in the more abundant mRNAs of the magnitude detected in sea urchin embryogenesis do not occur during amphibian oogenesis. RNA from oocytes of all stages directs 13Hllysine incorporation into product comigrating with calf thymus marker histones on both SDS and acid-urea gels. Translatable mRNA encoding hi&one-like proteins in vitro is detectable in the earliest, small stage 1, oocyte (Figs. 8 and 9). Our data also show that mRNA encoding histonelike proteins is found in the oocytes themselves and, thus, is not due primarily to the RNA content of the follicle cells or other ovarian tissues. Although it is clear that the onset of histone mRNA synthesis occurs very early in oogenesis, it is impossible to determine when, if at all, histone mRNA synthesis ceases during the growth of the oocyte, since the translation assay measures only the steady-state level of any mRNA. Furthermore, it is difficult to quantify, by cell-free translation, the amount of histone mRNA present at different stages of oogenesis, since RNA preparations obtained from various-stage oo-

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Development

--

a

b

c

d

e

f

FIG. 8. SDS gel electrophoresis of 13H]lysine-labeled translation product directed by RNA from Xenopus oocytes and ovary, as described in Table 1. Bars mark the positions of calf thymus histones. Product, 5 ~1, encoded by: (al early stage 1 oocyte RNA, (b) stage 2 oocyte RNA; (cl stage 4 oocyte RNA; (d) stage 6 oocyte RNA; (el whole ovary RNA; (f) no RNA added. Exposure: 10 days.

--

t-II

--

cytes contain different proportions of tRNA, 5s RNA and I-RNA; the effects of these RNAs on template activity are not well characterized. RNA obtained from early stage 1 oocytes shows significantly higher template activity per microgram of input RNA than do RNAs obtained from later-stage oocytes (Table 1). This is not surprising since very early oocytes contain a much higher proportion of poly(A)-containing RNA per microgram of total RNA than do the later stages (Rosbash and Ford, 1974). DISCUSSION

The Cell-Free

a

b

c

FIG. 9 Acid-urea gel e!lectrc bhoresis of [3H11ysine-labeled translation product directed by RNA fromXenopus oocytes, as described in Table 1. Bars mark positions of calf thymus histones. (al early stage 1 oocyte RNA (10 pl of product); (bl stage 4 oocyte RNA (10 pl of product); (c) whole ovary RNA (5 ~1 of product). Exposure: 14 days.

Translation

Assay

As measured by nucleic acid hybridization, the total sequence complexity of sea urchin egg mRNA, sea urchin embryo mRNA, or amphibian ovary mRNA is in the range of 2-3 x 10’ nucleotides, enough for some 12,000-18,000 different mRNA sequences (Davidson and Hough, 1971; Ros-

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DEVELOPMENTAL BIOLOGY VOLUME60, 1977

bash et al ., 1974; Anderson et al., 1976; Galau et al., 1974, 1976). Messenger RNA from a number of cell types, including sea urchin embryos, shows at least two distinct mRNA frequency classes in hybridization experiments. The major fraction of mRNA, the “abundant” or “prevalent” mRNA sequence class, consists of a relatively small number of different types of mRNA sequences, each present in very large numbers per cell. The minor fraction, the “complex” mRNA sequence class, comprises a very large number of different types of mRNA sequences, each present in only a few copies per cell. The prevalent mRNA makes up more than 95% of the cellular mRNA but, in general, represents less than 5% of the total mRNA complexity, sufficient to encode only a few hundred proteins (Galau et al., 1974; Bishop et al., 1974; Williams and Penman, 1975). We think it likely that the mRNA sequences detectable by cell-free translation represent, primarily, those mRNA species which are most prevalent in the population of translatable mRNA molecules, i.e., the abundant mRNA class. It is, however, by no means clear that all mRNAs are translated equally well in the wheat germ cell-free system. Certain mRNA sequences may exhibit intrinsically higher translational activity in vitro than others. Translational efficiencies in vitro may or may not reflect translational efficiencies in. uiuo. Various mRNAs do seem to exhibit different rates of protein synthesis both in uiuo and in uitro; these rates can be influenced by the presence of other RNA species (Lodish, 1971, 1974; Lodish and Jacobson, 1972; Jacobs-Lorena and Baglioni, 1972; McKeehan, 1974; Doe1 and Carey, 1976; Zilberstein et al., 1976; Wettenhall and Slobbe, 1976). While the cell-free system is limited and probably somewhat selective in the types of n-RNA sequences it can detect, it is capable of resolving at least some developmental changes in mRNA populations and yields information

which cannot be obtained as easily by hybridization experiments. In the studies reported here we have used the wheat germ cell-free protein-synthesizing system to demonstrate the presence of template-active RNAs in both poly(A)+ and poly(A)RNA obtained from sea urchin eggs and embryos and from amphibian (Xenopus ovaries and oocytes. We and Triturus) have also used the translation assay to screen for changes in abundant poly(A)+ and poly(A)- mRNAs in sea urchin embryogenesis and amphibian oogenesis. Comparison of in Vitro Translation Products Encoded by Poly(A) + and Poly(A) RNA of Sea Urchin Eggs and Amphibian Ovaries

Three general classes of mRNA have been described for a number of cell types: poly(A)+ mRNA, poly(A)- histone mRNA, and poly(A)- nonhistone mRNA (Adesnik et al., 1972; Milcarek et al., 1974; Nemer et al., 1974; Nemer, 1975; Fromson and Verma, 1976; Greenberg, 1976). Comparison of the cell-free translation products encoded by sea urchin egg RNA directly demonstrates the reality of template-active mRNA of each of these three general classes. Sea urchin egg poly(A)+ mRNA directs the cell-free synthesis of a large number of different polypeptide products. Sea urchin egg poly(A)- RNA is also very active in the cell-free system and directs incorporation into proteins corn&rating with marker histones, as well as a wide range of higher molecular weight nonhistone proteins. Sea urchin egg poly(A)+ and poly(A)RNA appear to encode both different and similar protein products when assayed by cell-free translation. Certain products seem to be encoded primarily by poly(A)+ mRNA, while others are mainly the product of poly(A)- mRNA. Many translation products, on the other hand, appear to be encoded by both poly(A)+ and poly(A)mRNA. Much of this overlap cannot be

RUDERMAN

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due to incomplete fractionation of poly(A)+ and poly(A)- RNAs, since the oligo(dT)cellulose fractionations were judged to be >95% complete by a number of criteria, including [3Hlpoly(U) hybridization, fractionation of Lytechinus pictus poly(A)histone mRNA, and fractionation of ribosomal RNA. At the present time, we cannot distinguish between comigration of nonidentical proteins encoded by poly(A)+ and poly(A)- RNAs and actual overlap in the coding sequences of the two RNA classes. However, adenylated and nonadenylated forms of a number of mRNAs, including those encoding globin (Cann et al., 1974), ovalbumin (Rosen et al., 1978, casein (Houbedine, 1976), protamines (Gedamu and Dixon, 1976), and histones (Levenson and Marcu, 1976; Ruderman and Pardue, submitted), have been demonstrated. Thus, it seems likely that some portion of sea urchin egg mRNA also exists in both poly(A)+ and poly(A)- forms. In contrast to the distribution of coding sequences in sea urchin egg poly(A)+ and poly(A)- RNA, the majority of the prevalent amphibian ovarian mRNA sequences appear to be polyadenylated. Both poly(A)+ and poly(A)- RNAs isolated from amphibian ovaries direct a substantial amount of protein synthesis in vitro. However, the only distinct detectable translation products encoded by Xenopus and Triturus ovary poly(A)- RNA are the histones. Our data demonstrate that amphibian ovary RNA lacks a component of abundant poly(A)- nonhistone n-RNA of the magnitude found in the sea urchin egg. The data certainly do not exclude the possibility that amphibian ovary poly(A)RNA contains, in addition to a substantial amount of histone mRNA, a component of rarer mRNA sequences. Such sequences would not be detectable in the translation assay under the conditions used here. It is possible that cell-free translation of prevalent nonhistone poly(A)) mRNAs of the amphibian ovary is masked by the presence of the substantial histone mRNA

mRNA

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component. This, however, is unlikely for two reasons. First, both unfractionated and poly(A)+ amphibian ovary mRNAs, which contain appreciable amounts of translatable histone mRNA, promote detectable incorporation into a large number of nonhistone translation products. Second, amphibian ovary poly(A)) mRNA which has been substantially depleted of histone mRNA by sucrose gradient sedimentation directs very little in vitro incorporation into higher molecular weight products (Ruderman, unpublished). Poly(A)+ and Poly(A)- Histone Sea Urchin Embryos

mRNA

in

Initial characterizations of histone mRNA showed that the bulk of histone mRNA is not polyadenylated in a number of cell types, including sea urchin (Lytechinus pictus) embryos and HeLa cells (Adesnik et al., 1972; Gruenstein et al., 1974; Nemer et al., 1974). Gruenstein and Schedl (1976) have shown by direct RNA sequence analysis that the smallest histone (H4) mRNA transcribed by Lytechinus cleavage-stage embryos lacks a poly(A) tract. Our results, obtained from analysis of the polypeptides encoded by Lytechinus morula polysomal poly(A)+ and poly(A)- RNAs in the cell-free translation system, show that most of the mRNA encoding the four lower molecular weight histones (H3, H2A, H2B, H4) is not polyadenylated. This is in direct agreement with previous findings that the bulk of Lytechinus embryonic histone mRNA is not polyadenylated (Gruenstein et al., 1974; Nemer et al., 1974; Gruenstein and Schedl, 1976). Surprisingly, however, a considerable amount of Lytechinus morula histone Hl is encoded in vitro by RNA fractionating with the poly(A)+ RNA class obtained by oligo(dT)-cellulose, as well as by poly(A)RNA. Examination of the translation products of poly(A)+ and poly(A)- RNAs isolated from another sea urchin species, Arbacia punctulata, suggests that, in this

64

DEVELOPMENTAL BIOLNY

species, some portions of all five of the major histone classes are encoded by poly(A)+ as well as by poly(A)- mRNA. The reasons for such species-specific differences are not known. Developmental Changes in the Poly(A)+ and Poly(A)- mRNAs during Early Sea Urchin Embryonic Development Several laboratories have described the changes in the relative amounts and size distributions of newly synthesized poly(A)+ mRNA, poly(A)- histone mRNA, and poly(A)- nonhistone mRNA which occur during sea urchin embryogenesis (Kedes and Gross, 1969a,b; Slater et al., 1973a,b; Wilt, 1973; Nemer et al., 1974; Nemer, 1975). Hybridization studies by Galau et al. (1976) demonstrate that there are significant differences in the sets of structural genes expressed at different stages of early sea urchin embryonic development, although large proportions of mRNA sequences are shared by all of the These hybridization stages examined. studies examine primarily the rare highcomplexity mRNA sequence component. Our data, obtained from cell-free translation of RNA, almost certainly concerns a very different component of mRNA, namely, the abundant mRNA class. The cell-free translation assay reveals that quantitative and, perhaps, qualitative changes in both poly(A)+ and poly(A)mRNAs occur during sea urchin embryonic development. Some poly(A)+ and poly(A)- mRNAs appear to be stage-specific, while others appear to be present throughout early embryogenesis, although in varying amounts. Of particular interest is the finding that the unfertilized egg contains an abundant poly(A)- mRNA, encoding a 70,000-80,000 MW protein of unknown function, which is not found in the morula-stage embryo. Recent experiments by Fryberg and Ruderman (1976) show that this particular mRNA is not polyadenylated following fertilization and that, as judged by the translation assay, this

VOLUME 60. 1977

mRNA persists until the 16-cell stage. The stage at which this mRNA is translated in vivo is not known. Quantitative and qualitative changes in histone mRNA during early sea urchin development and evidence for the transcriptional nature of the Hl switch have been discussed elsewhere (Rudermanet al., 1974; Arceciet al., 1976). Although sea urchin egg poly(A)mRNA encodes a wide range of nonhistone proteins in vitro, fewer such nonhistone products are detected in the translation product of morula or gastrula poly(A)mRNA (Fig. 6), and, with the exception of the histones, no striking changes in the spectra of proteins encoded by morula and gastrula poly(A)mRNA are detectable here. Our results suggest that sea urchin embryos, in contrast to eggs, contain relatively fewer prevalent nonhistone poly(A)mRNAs and add to the conclusions of Nemer et al. (1974) that newly synthesized nonhistone poly(A)mRNAs contribute less, at least quantitatively, to the embryonic program than do the poly(A)+ mRNA and histone mRNA components. The three different and complementary kinds of experimental evidence now available (patterns of RNA synthesis in vivo, molecular hybridization, and translation in vitro) demonstrate that stage-specific quantitative and qualitative changes in the expression of structural gene sequences encoding both prevalent and rare mRNA sequences occur during early sea urchin development, although a significant number of structural genes encoding both of these mRNA components is active throughout early embryogenesis. Poly(A)+ and Poly(A)Amphibian Oocytes

Histone

mRNA

in

The translation assay demonstrates that Xenopus and Triturus ovary RNA contains a rather considerable amount of histone mRNA. Virtually all of this histone mRNA is found in the oocytes and not in the follicle cells. Our results further indicate that a considerable amount of histone

RUDERMAN

mRNA oocyte. since it portion

AND

PARDUE

is present in the very early stage 1 These findings are not surprising, has previously been shown that a of the histones associated with Xenopus embryonic nuclear DNA is synthesized and stored during oogenesis (Adamson and Woodland, 1974). When RNA is fractionated into its poly(A)+ and poly(A)components by oligo(dT)-cellulose chromatography, both classes of RNA encode a substantial amount of material characterized as histones on the basis of comigration with calf thymus histones on both SDS and acidurea gels, differential lysine and methionine incorporation into Hl histone, characteristic migration of Hl histone on SDS and acid-urea gels, and differential acid extractability. Tryptic peptide analysis (Levenson and Marcu, 1976) of these histone-like products further supports the idea that histones are encoded by both poly(A)+ and poly(A)- amphibian ovary RNAs. However, because the translational efficiencies of histone mRNAs in the poly(A)+ and poly(A)- RNA preparations are not known, the relative amounts of poly(A)+ and poly(A)- histone mRNA in amphibian oocytes cannot be determined on the basis of the translation data alone. The detection of histone mRNA activity in the poly(A)+ RNA class obtained by oligo(dT)-cellulose chromatography suggests, but does not prove, the existence of poly(A)+ histone mRNA. More recent experiments (Ruder-man and Pardue, submitted) provide more direct evidence for the reality of poly(A)+ histone mRNA in amphibian oocytes. Our findings differ in some respects from those of Darnbrough and Ford (1976), reported that Xenopus who ovary poly(A)- RNA is not an efficient template for protein synthesis in vitro, and that the spectra of proteins encoded by poly(A)+ and poly(A)- are similar. Our data clearly show that amphibian ovary poly(A)RNA, as well as poly(A)+ RNA, is very active in directing protein synthesis in the

m.RNA in Early Development

65

wheat germ cell-free system, and that the relative proportions of histone and nonhistone mRNA in poly(A)+ and poly(A)RNAs are considerably different. Messenger RNA in Amphibian

Oogenesis

The results obtained in the study of template-active RNA is sea urchin embryogenesis show that the cell-free translation assay is capable of detecting developmental changes in abundant mRNAs. When this same approach is applied to the analysis of mRNA populations present in amphibian oocytes at different stages of oogenesis, no such striking changes in the spectra of template-active RNAs are observed. These results are in agreement with those of Darnbrough and Ford (1976). While our data do not rule out changes in the abundant mRNA sequences during amphibian oogenesis, they suggest that such changes are not as large as those occurring during sea urchin embryogenesis. It is, of course, possible and not unlikely that significant changes in rarer high-complexity mRNA sequence components do occur during oogenesis. We thank Thomas R. Cech, Ronald C. Herman, Richard C. Mulligan, and Bryan E. Roberts of this Institute for helpful discussions. Tom Humphreys generously provided facilities at the Marine Biological Laboratory, Woods Hole, for the initial experiments with Arbacia. We thank Hugh R. Woodland for carrying out the electrophoretic analysis presented in Fig. 4 and for helpful comments. This work was supported by a Jane Coffin Childs Postdoctoral Fellowship to J.V.R. and NSF Grant No. 7522581-BNS to M.L.P. REFERENCES E. D., and WOODLAND, H. R. (1974). Histone synthesis in early amphibian development: Histone and DNA syntheses are not co-ordinated.

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W., and DARall messenger RNA molecules (except histone messenger RNA) contain poly(A) sequences and that poly(A) has a nuclear function. J. Mol. Biol. 71, 21-30. ANDERSON, D. M., GALAU, G. A., BRITTEN, R. J., and DAVIDSON, E. H. (1976). Sequence complexity

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of the RNA accumulated in oocytes of Arbucia punctulutu. Develop. Biol. 51, 138-145. ARCECI, R. J., SENGER, D. R., and GROSS, P. R. (1976). The programmed switch in lysine-rich histone synthesis at gastrulation. Cell 9, 171-178. ATKINS, J. F., LEWIS, J. B., ANDERSON, C. W., and GESTELAND, R. F. (1975). Enhanced differential synthesis of proteins in a mammalian cell-free system by additions of polyamines. J. Biol. Chem. 250, 56884695. AVIV, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. PFOC. Nat. Acud. Sci. USA 69, 1408-1412. BACHAROVA, R., DAVIDSON, E. H., ALLFREY, V. G., and MIRSKY, A. E. (1966). Activation of RNA synthesis associated with gastrulation. Proc. Nut. Acad. Sci. USA 55, 358-365. BARTH, L. G., and BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in unconditioned medium. J. Embryol. Exp. Morphol. 7, 210-222. BENTTINEN, L., and COMB, D. (1971). Early and late histones during sea urchin development. J. Mol. Biol. 57, 355-358. BISHOP, J. O., MORTON, J. G., ROSBASH, M., and RICHARDSON, M. (1974). Three abundance classes in HeLa cell messenger RNA. Nature (London) 250, 199-204. BONNER, W., and LASKEY, R. A. (19741. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biothem. 46, 83-88. BROWN, D. D., and LITTNA, E.‘(196@. RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 8, 669687. BROWN, D. D., and LITTNA, E. (1966). Synthesis and accumulation of DNA-like RNA during embryogenesis ofxenopus laevis. J. Mol. Biol. 20, 81-94. CANN, A., GA~BINO, R., BANKS, J., and BANKS, A. (1974). Polyadenylate sequences and biologic activity of human globin messenger ribonucleic acid. J. Biol. Chem. 249, 7536-7540. COGNETTI, G., SPINELLI, G., and VIVOLI, A. (1974). Synthesis of histones during sea urchin embryogenesis. Biochim. Biophys. Actu 349, 447-455. DARNBROUGH, C., and FORD, P. J. (1976). Cell-free translation of messenger RNA from oocytes of Xenopus laevis. Develop. Biol. 50, 285-301. DAVIDSON, E. H. (19681. “Gene Activity in Early Development.” Academic Press, New York. DAVIDSON, E. H., and HOUGH, B. R. (1971). Genetic information in oocyte RNA. J. Mol. Biol. 56, 491506. DESTR~E, 0. H. J. (1975). “Histones in Development of Xenopus luevis.” Ph.D. Thesis, University of Amsterdam.

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RIJDERMAN AND PARDUE L. (1974). Messenger RNAs from individual histone proteins: Fingerprint analysis and in vitro translation. Cold Spring Harbor Symp. Quant. Biol. 38, 717-724. GRUENSTEIN, M., and SCHEDL, P. (1976). Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA. J. Mol. Biol. 104, 323-349. GURDON, J. B., and WOODLAND, H. R. (1969). The influence of the cytoplasm on the nucleus during cell differentiation, with special reference to RNA synthesis during amphibian cleavage. Proc. Roy. Sot. Ser. B 173, 99-111. HOGAN, B. L. M., and GROSS, P. R. (1971). The effect of protein synthesis on the entry of mRNA into the cytoplasm of sea urchin embryos. J. Cell Biol. 49, 692-701. HOUBEDINE, L. M. (1976). Absence of poly(A) in a large part of newly synthesized casein mRNA. FEBS Lett. 66, 110-113. HUMPHREYS, T. (1969). Efficiency of translation of messenger RNA before and after fertilization in sea urchins. Develop. Biol. 20, 435-458. HUMPHREYS, T. (1971). Measurements of messenger RNA entering polysomes upon fertilization of sea urchin eggs. Develop. Biol. 26, 201-208. JACOB, E., MALACINSKI, G., and BIRNSTIEL, M. L. (1976). Reiteration frequency of the histone genes in the genome of the amphibian, Xenopus laevis. Eur. J. Biochem. 69, 45-54. JACOBS-LORENA, M., and BACLIONI, C. (1972). Messenger RNA for globin in the post-ribosomal supernatant of rabbit reticulocytes. Proc. Nat. Acad. Sci. USA 69, 1425-1428. KEDES, L. H., and GROSS, P. R. (1969a). Synthesis and function of messenger RNA during early embryonic development. J. Mol. Biol. 42,559~575. KEDES, L. H., and GROSS, P. R. (1969b). Identification in cleaving embryos of three RNA species serving as templates for the synthesis of nuclear proteins. Nature (London) 223, 1335-1339. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227, 680-685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14Cin polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335341. LEVENSON, R., and MARCU, K. (1976). On the existence of polyadenylated histone messenger RNA in Xenopus laevis oocytcs. Cell 9, 311-322. LODISH, H. F. (1971). Alpha and beta globin messenger ribonucleic acid. J. Biol. Chem. 246, 71317138. LODISH, H. F. (1974). Model for the regulation of mRNA translation applied to haemoglobin synthesis. Nature (London) 251, 385-388. LODISH, H. F., and JACOBSON, M. (1972). Regulation of hemoglobin synthesis. J. Biol. Chem. 247,3622-

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