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RNA complementary to unique DNA sequences in normal and animalized sea urchin embryos
Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Exptirimental
Cell Research 92 (1975) 403-411
RNA COMPLEMENTARY TO UNIQUE DNA SEQUENCES IN NORMAL AND ANIMALIZED SEA URCHIN EMBRYOS Wm. R. ECKRERG and H. OZAKP Department of Zoology, Michigan State University, East Lansing, Mich. 48824, USA
SUMMARY
Early sea urchin developmentcan be experimentally manipulated so that abnormal embryos, called animalized embryos, develop with exaggeration of their ectodermal characteristicsand suppressionof their mesentodennalcharacteristics.In order to test whether this alteration in developmentinvolves changesin the pattern of embryonic genetranscription, embryonic RNA was exknined by RNA/DNA hybrid&ion. Rapidly i&&d RNA from both types of embryo hybridized to non-repetitive DNA readily, but much less readily to repetitive DNA. In order to determine the complexity of the RNA in normal and animalized embryos, purified radioactive non-repetitive DNA was incubated with a large excessof RNA. RNA was isolated from unfertilized eggsand from normal blastulae an&prism larvae and from animalized embryos of comparable ages. The complexity of transcription increasesduring the development of both normal and animalized embryos. Experiments in which RNAs isolated from two stageswere combined indicated that extensive homology exists between the populations, although some differences were detected between embryos of different ages as well as between normal and
animalized embryosof the sameage.This evidenceindicates that animalization involves alterations in the pattern of embryonic geneexpressionand that this abnormal developmentprovides a convenient experimental systemfor the study of gene regulation in embryonic development.
Sea urchin development is believed to be controlled by a coordination of two opposing gradients of morphogenetic influence, one maximal at the animal pole and the other maximal at the vegetal pole [29]. Disruption of this coordination through experimental manipulation can produce abnormal embryos with exaggeration of their ectodermal characteristics at the expense of their mesentodermal characteristics; such embryos are called animalized embryos. In its most severe form, animalization involves complete suppression of gut formation and hyperextension of the acronal stereocilia to cover much of the embryo [16]. RunnstrBm [30] hypothesized that the coordination of the animal-vegetal
gradients is mediated through the activity of the embryonic genome. This hypothesis is supported by the observation that the transcription inhibitor, actinomycin D, moderates the effects of animalizing and vegetalizing agents [21, 311. Analyses of proteins and RNA in animalized embryos are consistent with such an interpretation. Differences are observed between the electrophoretic patterns of newlysynthesized proteins of normal and animalized embryos [8]. Pirrone et al. [28] and O’Melia & Villee [27] have presented evidence that animalization involves an inhibition of the accumulation of ribosomal RNA (rRNA) which normally occurs concurrently with
* To whom reprint requests should be addressed.
gastrulation. In addition, in normal development the rate of uridine incorporation into
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RNA increases beginning at the blastula stage [12, 231, and an increase of the same magnitude but with a temporary delay accompanies animalization [12]. The present experiment were designed to test whether differences are found between the RNAs present in normal and animalized embryos. Such differences were observed by RNA/DNA hybridization. The results demonstrate that the pattern of embryonic transcription is altered in animalized embryos. MATERIALS AND METHODS Culture of embryos Gametes of the sea urchin, Strongylocentrotus purpurutus (Controlled Environments, Bellvue, Wash., or Pacific BioMarine, Venice, Calif.) were obtained by KC1 injection and fertilized. Embryos were cultured in artificial sea water (Aquarian Systems, Eastlake, Ohio), and animalization was effected by the addition of 5 x lo-* M ZnSO, to the sea water. Details of the culture methods have been published previously
1121. Reassociation of sperm DNA DNA. extracted from washed soerm by the method of Whiteley et al. [38], was disso&ed at a concentration of 1-2 mu/ml in 0.1 x SSC (0.15 M NaCl. 0.015 M Na citrate; pH 7.0), sheared in an Omnimixer at maximum speed for 5 min, dialysed against distilled water (3 changes? 50 vol each), and lyophiiized. DNA was then drssolved in 0.12 M PB (equimolar mono- and disodium phosphate) to the desired concentration (4-8 mg/ml) and sheared by sonic&ion to a length of 400-500 nudeotides as determined by the method of alkaline sedimentation analysis Il. 371. DNA was heat-denatured (10 min at -lOO°C) and allowed to reassociate at 60°C to various Cot values (moles nucleotide x set x 1-l) [6]. Single-stranded DNA was then degraded by S 1 nuclease. S 1 nuclease was prepared from Aspergillus oryzae a-amylase (Sigma) through step 4 of the method of Vogt [36], and concentrated by chromatography on a small column of DEAE-cellulose. Reassociated DNA was diluted to 150 pg/ml with 0.1 M NaCl, 0.001 M ZnSO,, 5 % glycerol, 0.03 M Na acetate, pH 4.6 and treated with 20-50 U/ml S 1 nuclease at 45°C for 30 mm. For non-radioactive DNA, samples were chilled. nrecinitated bv 5 % uerchloric acid. filtered through-a Millipore filter, type HA, and the absorbance (at 260 nm) of the filtrate (single-stranded material) was read. Radioactive samples were precipitated by TCA with 100 pg/ml BSA (bovine serum albumin) and the radioactivity in doubb-stranded material was determined as described above. Exptl Cell Res 92 (1975)
RNA/DNA
hybridization
in DNA excess
Prism stage embryos (48 h) and animalized embryos of the same age were labelled at a concentration of 10% v/v in artificial sea water containing 250 mg,/l streptomycin-sulfate (Sigma) and 3.3 ,&i/ml 3H-5uridine (22 Ci/mmole; Schwarz-Mann) for 30 min. Embryos were homogenized in 5 vol acetate-EDTA (0.01 M Na acetate, 0.01 M EDTA, pH 5.1) containing 0.1 SDS and 0.1 % bentonite, and RNA was extracted at 60°C by shaking with an equal volume of phenol. The aqueous phase was reextracted twice with phenol at 0°C and the RNA was precipitated by ethanol. Radioactive RNA was then digested at 37°C for 1 h with 50 ,ug/ml DNase (RNase-free, Worthington). After additional deproteinization with pronase (50 fig/ml for 1 h at 37°C) and phenol the RNA was precipitated by ethanol. The RNA was then dissolved in 0.12 M PB and sheared to annrox. 6S by sonication. Final preparations had specific activities of approx. 1 000 dpm/pg and the radioactivity was greater than 99.5 % alkali-labile. The RNA specific activities from normal and animalized embrvos were identical. Sheared radioactive RNA (1 pg) was added to a lOO-fold excess of sperm DNA in 0.12 M PB containing 0.1 % SDS. After heat denaturation (100°C for 5 min), the mixtures were incubated at 60°C to the desired DNA Cot, diluted to 5 ml in 0.24 M PB and divided in half. One half was incubated with 20 pg/ml RNase (bovine pancreas, Worthington) for 20 min at 37°C. RNase had been heated previously at 80°C for 10 min to destroy the DNase activity. The other half was treated similarly but without RNase. Mixtures were precipitated by TCA, and the radioactivity was determined as above.
Preparation of radioactive non-repetitive DNA A 1% susoension of embryos was labelled from fertilization-to early gastrula &age (36 h) with 2,Li/ml *H-methyl-thymidine (16.7 Ci/rrmrole, Schwarz-Mann). Embryos were washed twice in 0.53 M NaCl, 0.53 M KC1 (19: 1). twice in 1 M dextrose. and once in SSC. The final ‘pellet was suspended in 30 vol SSC and homogenized by two passages through a no. 20 gauge hypodermic needle. The homogenate was mixed with an equal volume of 2 M sucrose and centrifuged at 15 000 g for 30 min. The nuclear pellet was suspended in 5 ml 0.1 M EDTA, 0.04 M Tris-HCl, pH 8.2, heated at 60°C for 10 min, made 1 % in SDS and 1 M in NaClO,, shaken with an equal volume of chloroform-isoamyl alcohol (24 : 1) and precipitated by ethanol. DNA was dissolved in 5 ml 0.1 x SSC and incubated with 50 uelml heattreated RNase at 37°C for 30 min. DNA was made to 0.1 M Tris, pH 9.0, 0.1 M NaCl and 1 % SDS, shaken with an equal volume of phenol and precipitated by ethanol. The final ureparations had specific activities of about 120 000 dpm&g. Purified DNA was then dissolved in 0.12 M PB and sheared. An aliauot was denatured and allowed to reassociate in a iarge excess of non-radioactive snerm DNA. The kinetics of reassociation of radioactive and non-radioactive DNA were the same. Radioactive DNA was then heat-denatured and incubated to Cot 30. Singlestranded material was .
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RNA in abnormal sea urchins
405
Fig. 1, (a) Normal prism larva of Sfrongylocentrotus purpuratus. Note the regionally-differentiated gut and the skeletal spicules. ( x 300); (b) animalized embryo of the same age and from the same batch as the embryo shown in (a) but raised in the presence of 5 x 1O-4M ZnSO, added to the sea water. Note that the outer diameter of the animalized embryo is greater due to the fact that the presumptive endodermal cells are a part of the ectoderm. Also note the aggregated descendants of the primary mesenchyme cells in the blastocoel and the absence of skeletal spicules. ( x 300). purified by HAP (hydroxyapatite) chromatography, heat-denatured. reincubated to C.t 30. and repurified from HAP. The purified non-repetitive DNA was dialvsed against distilled water. lvonhilized, and dissolved in a small volume of’ 0.12- M PB: To check its purity, an aliquot of this radioactive non-repetitive DNA was heat-denatured and allowed to reassociate with a large excess of total sperm DNA.
RNA/DNA
hybridization
in RNA excess
Non-radioactive RNA was extracted from unfertilized eggs and from normal blastulae (24 h) and prism larvae (48 h) and from animalized embrvos of comparable ages as described above. The isolated RNA was further purified by DNase digestion and cetyltrimethyl ammonium bromide precipitation [2], sheared, dialysed against distilled water, lyonhilized, and dissolved in 0.5 M NaCl, 0.001 M EDTA, 0102 M Tris-HCI, pH 7.4, containing 0.1 % SDS (after [20]). Heat-denatured radioactive non-repetitive DNA was incubated with an excess of RNA in the above buffer at 60°C. After incubation the mixtures were diluted to 150 .walrnl -, RNA in the Sl nuclease buffer and divided in half. Half was treated with S 1 nuclease as described above, and the other half was treated similarly but without Sl nuclease. All incubations were to the same equivalent COfwith respect to DNA (2.14), to eliminate possible variability in data due to DNA renaturation. In order to determine the extent of DNA renaturation and the possible contamination of the RNA RNA preparations with DNA, RNA which had been hydrolyzed (0.3 N NaOH, 18 h at 37°C) was incubated with the DNA as above. Less than 1.1 % of the input radioactivity was S 1-nuclease-insensitive and this value was subtracted from the observed percents reassociated. Since this value was not greater than the value observed at that Cot in the excess of total
sperm DNA (fig. 2), the RNA preparations were not contaminated with DNA.
Thermal stability of hybrids Hybrids were diluted to 0.1 M NaCl and their thermal stability was determined by raising the temperature of the solution from 60 to 100°C in increments of 5°C. After equilibration for 5 min at each 5°C increment, an aliquot was removed and digested with Sl nuclease, and the S 1 nuclease-insensitive radioactivity was determined as above.
RESULTS Embryonic development
Animalized embryos develop into large thinwalled motile blastula-like structures covered with stereocilia and endowed with a small aggregate of cells in the blastocoel near the vegetal pole. Such embryos completely lack a gut at a stage when the gut of normal embryos is regionally differentiated (fig. 1). The viability of such embryos is equal to that of non-fed control embryos. Templates for rapidly-labelled heterogeneous RNA
Assayed by Sl nuclease the genome of the sea urchin appears to contain at least 30 % repeated sequences, and the non-repetitive component reassociates with a 8 Cot of Exptl Cell Res 92 (1975)
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Eckberg and Ozaki
I 0.1
I I
I IO
I 100
I 1000
I IO 000
Fig. 2. Abscissa: C,t; ordinate: % Sl nuclease insensitive. Reassociation kinetics of sea urchin suerm DNA assayed by Sl nuclease (0). Reassociation kinetics of radioactive non-repetitive gastrula DNA in the presence of a 1 000-fold excess of total sperm DNA assayed by S 1 nuclease ( l ).
during the development of normal and animalized embryos, RNA/DNA hybridization was carried out using purified radioactive non-repetitive DNA. That this fraction of DNA is essentially free of repetitive sequences is shown by its hybridization kinetics with total sperm DNA (fig. 2). The technique employed for saturation hybridization involved the incubation of labelled DNA of high specific activity with a large excess of non-radioactive RNA. At saturation, doubling of the RNA concentration or time of incubation results in no further increase in the per cent hybridization. A typical saturation curve is presented in fig. 4 and shows that, in the case of normal prism larvae, saturation is approached when 7.7 % of the DNA is hybridized. Assuming asymmetric transcription, this corresponds to approx. 15.4% of the non-repetitive DNA. The apparent saturation values obtained at all stagestested are listed in table 1. It should be pointed out that since saturation is never actually reached the data represent minimal estimates of the genomic information present. To compare the transcripts present at different stagesof the development of normal d
approx. 800 (fig. 2). In order to determine whether rapidly-labelled RNA is transcribed from repetitive or non-repetitive DNA sequencesand whether the relative transcription from repetitive and non-repetitive DNA sequences is different between normal and animalized embryos, RNA/DNA hybridization was carried out in DNA excess. RNAs from normal prism larvae and from animalized embryos of the sameageboth reassociated primarily with non-repetitive DNA (fig. 3). At a Cot of 30, 7-10s of the material was RNase-insensitive, but since 3-4s was RNase-insensitive at zero-time, the actual amount hybridized is probably on the order of 5 % at this Cot value. The 9 Cot for the reaction of the RNA with non-repetitive DNA is within a factor of 2 of that for the DNA reassociation. The scatter of the data precludes a more precise determination of the hybridization kinetics. Genomic representation in RNA of normal and animalized embryos
In order to assay directly for the amount of genomic information expressed in RNA Exptl Cell Res 92 (1975)
0, ,
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I 0.1
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I 100
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I’
0 so
1 0.01
I 10000
Fig. 3. Abscissa: DNA C,,t; ordinate: % ribonucleaseinsensitive. Kinetics of hybridization of 30 mm pulse-labelled RNA from 0, normal prism larvae; 0, animalized embryos of the same age with a lOO-fold excess of total sperm DNA.
RNA in abnormal sea urchins
Table 1. Per cent of non-repetitive
DNA hybridized to RNA from various developmental stages of normal and animalized embryos
I” a-
I
0
IO
I
I
401
I a-
RNA pg/ml x h x 1O-4 Stage Unfertilized egg Blastula (24 h) Prism(48 h) Animalized (24 h) Animalized (48 h) Blastula + animalized (24 h)a Prism t animalized (48 h)’ Prism + blastulaa Animalized (24 h) + animalized (48 h)”
24 4.4 6.4 6.9 E 810 8.9 7.9
48 4.5 7.3 7.7 7.6 8.1
20
I
I
30
40
50
Fig. 4. Abscissa: RNA ,ug/ml x h x lo-$ ordinate: % DNA S 1 nuclease-insensitive. Saturation of radioactive non-repetitive DNA with RNA from 0, normal prism larvae; l , animalized embryos of the same age. RNA/DNA ratios ranged up to 10 000: 1. All incubations were to the same equivalent DNA Cot (2.14) to eliminate possible variation in results due to DNA renaturation.
7.1
a In additive experiments the concentration x time factor for each RNA reactant was 24 x lo* ,ug/ml x h.
and animalized embryos, additive experiments were performed in which the DNA was hybridized with RNAs from two different stages simultaneously. The difference between the arithmetic sum of the saturation values of each RNA preparation separately and that obtained in the additive experiments represents the extent of homology between the RNA populations. The results of these experiments are found in table 1. Since the concentration x time factor for each RNA reactant was 24 x lo4 pg/ml x h, the total concentration x time factor was twice that for RNA speciescommon to the two preparations. Thus an exact quantitative estimate of the homology between the populations cannot be made. However, by comparing the values from the additive experiment with those from the longer and shorter incubations, upper and lower limits can be placed on the extent of homology. For the prism + blastula experiment, 6.4 + 6.9-7.9 = 5.4, the lower limit of the per cent of the genome represented in both the normal blastula and prism. The upper limit is 7.3+7.7-7.9=7.1. Since (5.4/6.0)=0.79 and
(7.1/7.7) =0.92 we conclude that between 79 and 92% of the sequences present in the prism are also present in the blastula. By similar calculations, between 78 and 95 % of the non-repetitive DNA transcripts of 24 h blastulae are also present in 24 h animalized embryos; between 78 and 90% of the nonrepetitive DNA transcripts of the 48 h prism larva are also present in 48 h animalized embryos, and essentially 100% of the nonrepetitive transcripts in 24 h animalized embryos are retained in 48 h animalized embryos. Thus the difference between normal and animalized embryos of the same age is of the same magnitude as the difference between normal blastulae and prism larvae. Thermal stability of RNA/DNA
hybrids
The thermal stability of radioactive nonrepetitive DNA/total sperm DNA hybrids and that of radioactive non-repetitive DNA/ RNA hybrids is given in fig. 5. The DNA/ DNA hybrids melt very sharply with a T, of 78”C, whereas the RNA/DNA hybrids melt sharply with a T,,, of 71°C. The sharp melting profile, indicating that all hybrids are of equal stability, is characteristic of non-repetitive RNA/DNA hybrids and not of repetitive RNA/DNA hybrids. The observed depression is due, at least in part, to Exptl Cell Res 92 (1975)
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Eckberg and Ozaki
60
40
20
0 6
Fig. 5. Abscissa: temp. (“C); ordinate: % S 1 nucleaseinsensitive radioactivity released. Thermal denaturation profiles of radioactive nonrepetitive DNA/total sperm DNA hybrids (0); DNA/RNA hybrids (0), in 0.1 M Na+, assayed by Sl nuclease. T, of DNA/DNA hybrids is 78°C; T,,, of RNA/DNA hybrids is 71°C.
short hybrid regions [15, 331. The maximum length of the hybrids is about 200 nucleotide pairs as limited by the size of the RNA molecules hybridized. With the above considerations and the fact that well-matched bacterial RNA/DNA hybrids can melt with approximately a 5°C drop in T,,, [4], we conclude that very little mismatch is present, and thus that near locus specificity has been achieved. DISCUSSION RNA transcripts have been studied in animalized and normal sea urchin development by homology with various regions of the genome by RNA/DNA hybridization. Sucrose gradient analysis demonstrates that most of the radioactivity incorporated by prism larvae and by animalized embryos ExpfI Cell Res 92 (1973)
during a short pulse label is incorporated into heterogeneously sedimenting high molecular weight material, most likely heterogeneous nuclear RNA (hnRNA) [12]. In order to determine whether this material is synthesized from repetitive or non-repetitive DNA sequences, this material was hybridized to DNA in an excessof DNA. When RNA is present in trace quantities in a DNA/DNA reassociation mixture, the RNA will hybridize to the DNA and the kinetics of hybrid formation will reflect the degree of reiteration of the homologous DNA in the genome. For the interpretation of such experiments, the DNA excess must be large; at least 70-loo-fold [3]. In the experiments reported here, the DNA excess was loo-fold, but it should be mentioned that less than 10% of the cellular RNA in the reaction mixture is the hnRNA in which nearly all the label is found. Thus it is likely that the DNA is in at least a 1000-fold excess over hybridizable radioactive RNA sequences. Under these conditions, the reaction should give an accurate picture of the reiteration frequency of the genes transcribing hnRNA in normal and animalized seaurchin embryos. From these data we conclude that the representation in hnRNA of the repetitive sequences of the sea urchin genome is small. Smith et al. [33], using purified gastrula hnRNA and a DNA excessof over 100 OOOfold obtained similar results. The nature of the zero-time RNase-insensitive material was not further studied, but this fraction may correspond to the fraction of hnRNA known to be double-stranded [18]. This material could be rendered RNase-insensitive either through intramolecular base pairing or through its rapid hybridization to DNA 1171. In order to assay for the complexity of RNA transcripts present at different stages of development, RNA/DNA hybridization
RNA in abnormal sea urchins
was carried out using purified non-repetitive DNA in a large excess of RNA. Under such conditions, the contribution of the DNA to the rate of hybridization may be ignored and the reaction is pseudo firstorder. In experiments using total cellular RNA the actual concentration of hybridizable RNA sequencesis unknown since most of the cellular RNA is ribosomal (rRNA) and transfer RNA (tRNA) which do not bind to non-repetitive DNA [7, 25, 341. Nevertheless, saturation plateaus can be determined and the extent of homology between two populations of RNA can be estimated. Although purified non-repetitive DNA fractions will contain, in the limit, one copy of each repetitive DNA sequence, all or nearly all the observed hybridization must be to non-repetitive sequences, since this DNA is vastly enriched for non-repetitive sequences, and nearly all rapidly-labelled heterogeneous RNA ([33] and fig. 3) and all messenger RNA [14] hybridize to nonrepetitive DNA. The data of table 1 demonstrate that during the development of both normal and animalized embryos the complexity of transcription increases and that stage-specific patterns are observed. Transcripts are present at each stage tested which are unique to that particular stage. It is of particular interest that relatively large differences are observed between the unique DNA sequence transcripts of normal and animalized embryos of the same age. The data of table 1 also demonstrate that the homology between the RNAs derived from non-repetitive DNA of different stages of development is similar to that of RNAs derived from repetitive DNA sequences[38]. In addition it may be noted that the saturation levels obtained at different stages of development correspond roughly to the complexity of transcription at analogous
409
stagesin the development of other organisms. Davidson & Hough [lo] saturated 0.9 % of the non-repetitive portion of the Xenopus laevis genome with unfertilized egg RNA. Since the Xenopus genome is about 4 times as large as that of the sea urchin, the complexity of the stored RNA messagesin the unfertilized Xenopus egg is similar to that in the unfertilized sea urchin egg. Davis & Wilt [ll] have reported that the unfertilized egg of the marine worm, Urechis caupo, which has a genome slightly larger than that of the sea urchin, contains RNA complementary to 4.3 % of its non-repetitive DNA. Thus the RNA complexity in the unfertilized egg of Urechis is nearly the same as that in the unfertilized sea urchin egg. Schultz et al. [32] have shown that 1.8 % of the nonrepetitive portion of the rabbit genome is represented in RNA in preimplantation blastocysts and that 2.5 % is represented in post-implantation blastocysts which have begun to differentiate. Since the rabbit genome is about 4 times as large as that of the seaurchin, the actual amount of information in these stagesof rabbit embryos is very close to that present in sea urchin embryos at the analogous stages. Similar values have been reported for early mouse development [9]. Such results are not surprising as the embryos of all these organisms are carrying out similar activities at analogous stages;e.g. mitosis, primary germ layer formation, maintenance of metabolism, etc. The high degree of homology between the RNAs of different stagesof development may reflect great stability of messages.The unfertilized egg contains messagessufficient to support development through the blastula stage [35]. It is possible that some of these messagesare retained for long periods during development. It is believed that during development messagesare transcribed long before they are to be used by the embryo to support Exptl Cell Res 92 (1975)
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differentiation [13, 35, 381. The presence of such long-lived messageswould lead to the observed high degree of homology between the RNAs present at different stages of development. The half-life of newly-synthesized mRNA in sea urchin embryos has been reported to be about 75 min [5]. Recent measurementshave indicated that in cultured cells the half-life of mRNA is much longer, however [24]. The existence of even a small fraction of very long-lived mRNA in sea urchin embryos might provide the results obtained. Alternatively, most of the messages in RNA in the early sea urchin embryo may not be concerned with differentiation, but rather with activities common to all cells. It has long been known that sea urchin development can be altered by various operative and chemical methods. Various types of experiments have implicated the genome in the control of sea urchin development and its experimental alteration. Treatment with actinomycin D reduced the animalization of isolated animal half embryos [22] and that of whole embryos chemically animalized [19, 211.Animalization also alters the protein synthetic pattern. Esterase isozyme activities which appear in normal pluteus larvae do not appear in animalized embryos of the same age [26], and there are differences between the newly-synthesized proteins of normal and animalized embryos [8]. The present results demonstrate that alterations in the pattern of embryonic gene expression do occur in animalization, and therefore that these alterations can be conveniently induced in embryos merely by altering their ionic environment. Taken together with the results cited above, they suggest that alterations in genetic activity may, indeed, lead to the developmental abnormality known as animalization. This research was supported in part by a Grant-in-Aid of research from the Society of the Sigma Xi to ExptI Cell Res 92 (1975)
W. R. E. and by a Bio-Medical Sciences Support grant from Michigan State University to H. 0.
REFERENCES 1. Abelson, J & Thomas, C A, J mol biol 18 (1966) 262.
2. Bellamy, A R & Ralph, R K, Meth enzymol 12B (1968) 156. 3. Bishou. J 0. Biochem i 126 (1972) 171. 4. Bolton; E T & McCarthy,‘ B J, J mol biol 8 (1964) 201. 5. Brandhorst, B P & Humphreys, T, J cell biol 53 (1972) 474. 6. Britten, R J & Kohne, D E, Science 161 (1968) 529. 7. Brown, D D & Weber, C S, J mol biol 34 (1968) 661. 8. Carroll, A G, Eckberg, W R & Ozaki, H, Exptl cell res 90 (1975) 328. 9. Church, R B & Brown, I R, Nucleic acid hybridization in the study of cell differentiation (ed H Ursprung) p. 11. Springer, New York (1972). 10. Davidson; E H & Hough, B R, J mol biol 56 (1971) 491. 11. Davis, F C &Wilt, F H, J cell biol 59 (1973) 72a. 12. Eckberg, W R & Ozaki, H, Exptl cell res 73 (1972) 177. 13. Giudice, G, Mutolo, V & Donatuti, G, Wilhelm Roux’ Arch Entwicklungsmech Organ 161 (1968) 118. 14. Goldberg, R B, Galau, G A, Britten, R J & Davidson, E H, Proc natl acad sci US 70 (1973) 3516. 15. Hayes, F N, Lilly, E H, Ratliff, R L, Smith, D A & Williams, D L, Biopolymers 9 (1970) 1105. 16. Horstadius, S, Biol rev 14 (1939) 132. 17. Jelinek, W & Darnell, J E, Proc natl acad sci US 69 (1972) 2537. 18. Kronenberg, L H & Humphreys, T, Biochemistry 11 (1972) 2020. Lallier, R, Compt rend acad sci 257 (1963) 2159. :;: Leong, J-A, Garapin, A-C, Jackson, N, Fanshier, L, Levinson, W & Bishop, J M, J viral 9 (1972) 891. 21. Markman. B & Runnstrom. J. Exotf cell res 31 (1963) 615. 22. - Wilhelm Roux’ Arch Entwickhmgsmech Organ 165 (1970) 1. 23. Mizuno, S, Whiteley, H R & Whiteley, A H, Differentiation 1 (1973) 339. 24. Murphy, W & Attardi, G, Proc natl acad sci US 70 (1973) 115. 25. Mutolo, V & Giudice, G, Biochim biophys acta 149 (1967) 291. 26. O’Melia, A F, Exptl cell res 73 (1972) 469. 27. O’Melia A F & Villee. C A. Exntl cell res 72 (1972) 276. 28. Pirrone, A M, Sconzo, G, Mutolo, V & Giudice, G, Wilhelm Roux’ Arch Entwicklungsmech Organ 164 (1970) 222. 29. Runnstrom, J, Wilhelm Roux’ Arch Entwicklungmech Organ 129 (1933) 442. z,
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RNA in abnormal sea urchins 411 30. - Exptl biol med 1 (1967) 52. 31. Runnstrom, J & Markman, B, Biol bull 130 (1966) 404. 32. Schultz, G, Manes, C & Hahn, W E, Biochem genet 9 (1973) 247. 33. Smith, M J, Hough, B R, Chamberlin, M E & Davidson, E H, J mol biol 85 (1974) 103. 34. Sy, J & McCarty, K S, Biochim biophys acta 199 (1970) 86.
35. Tyler, .A, Dev biol, suppl. 1 (1967) 170. 36. Vogt, V M, Eur j biochem 33 (1973) 192. 37. Wetmur, J G & Davidson, N, J mol biol 31 (1968) 349. 38. Whiteley, H R, McCarthy, B J & Whiteley, A H, Dev biol 21 (1970) 216. Received October 22, 1974 Revised version received December 2, 1974
Exptl Cell Res 92 (1975)
Exptirimental
Cell Research 92 (1975) 403-411
RNA COMPLEMENTARY TO UNIQUE DNA SEQUENCES IN NORMAL AND ANIMALIZED SEA URCHIN EMBRYOS Wm. R. ECKRERG and H. OZAKP Department of Zoology, Michigan State University, East Lansing, Mich. 48824, USA
SUMMARY
Early sea urchin developmentcan be experimentally manipulated so that abnormal embryos, called animalized embryos, develop with exaggeration of their ectodermal characteristicsand suppressionof their mesentodennalcharacteristics.In order to test whether this alteration in developmentinvolves changesin the pattern of embryonic genetranscription, embryonic RNA was exknined by RNA/DNA hybrid&ion. Rapidly i&&d RNA from both types of embryo hybridized to non-repetitive DNA readily, but much less readily to repetitive DNA. In order to determine the complexity of the RNA in normal and animalized embryos, purified radioactive non-repetitive DNA was incubated with a large excessof RNA. RNA was isolated from unfertilized eggsand from normal blastulae an&prism larvae and from animalized embryos of comparable ages. The complexity of transcription increasesduring the development of both normal and animalized embryos. Experiments in which RNAs isolated from two stageswere combined indicated that extensive homology exists between the populations, although some differences were detected between embryos of different ages as well as between normal and
animalized embryosof the sameage.This evidenceindicates that animalization involves alterations in the pattern of embryonic geneexpressionand that this abnormal developmentprovides a convenient experimental systemfor the study of gene regulation in embryonic development.
Sea urchin development is believed to be controlled by a coordination of two opposing gradients of morphogenetic influence, one maximal at the animal pole and the other maximal at the vegetal pole [29]. Disruption of this coordination through experimental manipulation can produce abnormal embryos with exaggeration of their ectodermal characteristics at the expense of their mesentodermal characteristics; such embryos are called animalized embryos. In its most severe form, animalization involves complete suppression of gut formation and hyperextension of the acronal stereocilia to cover much of the embryo [16]. RunnstrBm [30] hypothesized that the coordination of the animal-vegetal
gradients is mediated through the activity of the embryonic genome. This hypothesis is supported by the observation that the transcription inhibitor, actinomycin D, moderates the effects of animalizing and vegetalizing agents [21, 311. Analyses of proteins and RNA in animalized embryos are consistent with such an interpretation. Differences are observed between the electrophoretic patterns of newlysynthesized proteins of normal and animalized embryos [8]. Pirrone et al. [28] and O’Melia & Villee [27] have presented evidence that animalization involves an inhibition of the accumulation of ribosomal RNA (rRNA) which normally occurs concurrently with
* To whom reprint requests should be addressed.
gastrulation. In addition, in normal development the rate of uridine incorporation into
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RNA increases beginning at the blastula stage [12, 231, and an increase of the same magnitude but with a temporary delay accompanies animalization [12]. The present experiment were designed to test whether differences are found between the RNAs present in normal and animalized embryos. Such differences were observed by RNA/DNA hybridization. The results demonstrate that the pattern of embryonic transcription is altered in animalized embryos. MATERIALS AND METHODS Culture of embryos Gametes of the sea urchin, Strongylocentrotus purpurutus (Controlled Environments, Bellvue, Wash., or Pacific BioMarine, Venice, Calif.) were obtained by KC1 injection and fertilized. Embryos were cultured in artificial sea water (Aquarian Systems, Eastlake, Ohio), and animalization was effected by the addition of 5 x lo-* M ZnSO, to the sea water. Details of the culture methods have been published previously
1121. Reassociation of sperm DNA DNA. extracted from washed soerm by the method of Whiteley et al. [38], was disso&ed at a concentration of 1-2 mu/ml in 0.1 x SSC (0.15 M NaCl. 0.015 M Na citrate; pH 7.0), sheared in an Omnimixer at maximum speed for 5 min, dialysed against distilled water (3 changes? 50 vol each), and lyophiiized. DNA was then drssolved in 0.12 M PB (equimolar mono- and disodium phosphate) to the desired concentration (4-8 mg/ml) and sheared by sonic&ion to a length of 400-500 nudeotides as determined by the method of alkaline sedimentation analysis Il. 371. DNA was heat-denatured (10 min at -lOO°C) and allowed to reassociate at 60°C to various Cot values (moles nucleotide x set x 1-l) [6]. Single-stranded DNA was then degraded by S 1 nuclease. S 1 nuclease was prepared from Aspergillus oryzae a-amylase (Sigma) through step 4 of the method of Vogt [36], and concentrated by chromatography on a small column of DEAE-cellulose. Reassociated DNA was diluted to 150 pg/ml with 0.1 M NaCl, 0.001 M ZnSO,, 5 % glycerol, 0.03 M Na acetate, pH 4.6 and treated with 20-50 U/ml S 1 nuclease at 45°C for 30 mm. For non-radioactive DNA, samples were chilled. nrecinitated bv 5 % uerchloric acid. filtered through-a Millipore filter, type HA, and the absorbance (at 260 nm) of the filtrate (single-stranded material) was read. Radioactive samples were precipitated by TCA with 100 pg/ml BSA (bovine serum albumin) and the radioactivity in doubb-stranded material was determined as described above. Exptl Cell Res 92 (1975)
RNA/DNA
hybridization
in DNA excess
Prism stage embryos (48 h) and animalized embryos of the same age were labelled at a concentration of 10% v/v in artificial sea water containing 250 mg,/l streptomycin-sulfate (Sigma) and 3.3 ,&i/ml 3H-5uridine (22 Ci/mmole; Schwarz-Mann) for 30 min. Embryos were homogenized in 5 vol acetate-EDTA (0.01 M Na acetate, 0.01 M EDTA, pH 5.1) containing 0.1 SDS and 0.1 % bentonite, and RNA was extracted at 60°C by shaking with an equal volume of phenol. The aqueous phase was reextracted twice with phenol at 0°C and the RNA was precipitated by ethanol. Radioactive RNA was then digested at 37°C for 1 h with 50 ,ug/ml DNase (RNase-free, Worthington). After additional deproteinization with pronase (50 fig/ml for 1 h at 37°C) and phenol the RNA was precipitated by ethanol. The RNA was then dissolved in 0.12 M PB and sheared to annrox. 6S by sonication. Final preparations had specific activities of approx. 1 000 dpm/pg and the radioactivity was greater than 99.5 % alkali-labile. The RNA specific activities from normal and animalized embrvos were identical. Sheared radioactive RNA (1 pg) was added to a lOO-fold excess of sperm DNA in 0.12 M PB containing 0.1 % SDS. After heat denaturation (100°C for 5 min), the mixtures were incubated at 60°C to the desired DNA Cot, diluted to 5 ml in 0.24 M PB and divided in half. One half was incubated with 20 pg/ml RNase (bovine pancreas, Worthington) for 20 min at 37°C. RNase had been heated previously at 80°C for 10 min to destroy the DNase activity. The other half was treated similarly but without RNase. Mixtures were precipitated by TCA, and the radioactivity was determined as above.
Preparation of radioactive non-repetitive DNA A 1% susoension of embryos was labelled from fertilization-to early gastrula &age (36 h) with 2,Li/ml *H-methyl-thymidine (16.7 Ci/rrmrole, Schwarz-Mann). Embryos were washed twice in 0.53 M NaCl, 0.53 M KC1 (19: 1). twice in 1 M dextrose. and once in SSC. The final ‘pellet was suspended in 30 vol SSC and homogenized by two passages through a no. 20 gauge hypodermic needle. The homogenate was mixed with an equal volume of 2 M sucrose and centrifuged at 15 000 g for 30 min. The nuclear pellet was suspended in 5 ml 0.1 M EDTA, 0.04 M Tris-HCl, pH 8.2, heated at 60°C for 10 min, made 1 % in SDS and 1 M in NaClO,, shaken with an equal volume of chloroform-isoamyl alcohol (24 : 1) and precipitated by ethanol. DNA was dissolved in 5 ml 0.1 x SSC and incubated with 50 uelml heattreated RNase at 37°C for 30 min. DNA was made to 0.1 M Tris, pH 9.0, 0.1 M NaCl and 1 % SDS, shaken with an equal volume of phenol and precipitated by ethanol. The final ureparations had specific activities of about 120 000 dpm&g. Purified DNA was then dissolved in 0.12 M PB and sheared. An aliauot was denatured and allowed to reassociate in a iarge excess of non-radioactive snerm DNA. The kinetics of reassociation of radioactive and non-radioactive DNA were the same. Radioactive DNA was then heat-denatured and incubated to Cot 30. Singlestranded material was .
I.
RNA in abnormal sea urchins
405
Fig. 1, (a) Normal prism larva of Sfrongylocentrotus purpuratus. Note the regionally-differentiated gut and the skeletal spicules. ( x 300); (b) animalized embryo of the same age and from the same batch as the embryo shown in (a) but raised in the presence of 5 x 1O-4M ZnSO, added to the sea water. Note that the outer diameter of the animalized embryo is greater due to the fact that the presumptive endodermal cells are a part of the ectoderm. Also note the aggregated descendants of the primary mesenchyme cells in the blastocoel and the absence of skeletal spicules. ( x 300). purified by HAP (hydroxyapatite) chromatography, heat-denatured. reincubated to C.t 30. and repurified from HAP. The purified non-repetitive DNA was dialvsed against distilled water. lvonhilized, and dissolved in a small volume of’ 0.12- M PB: To check its purity, an aliquot of this radioactive non-repetitive DNA was heat-denatured and allowed to reassociate with a large excess of total sperm DNA.
RNA/DNA
hybridization
in RNA excess
Non-radioactive RNA was extracted from unfertilized eggs and from normal blastulae (24 h) and prism larvae (48 h) and from animalized embrvos of comparable ages as described above. The isolated RNA was further purified by DNase digestion and cetyltrimethyl ammonium bromide precipitation [2], sheared, dialysed against distilled water, lyonhilized, and dissolved in 0.5 M NaCl, 0.001 M EDTA, 0102 M Tris-HCI, pH 7.4, containing 0.1 % SDS (after [20]). Heat-denatured radioactive non-repetitive DNA was incubated with an excess of RNA in the above buffer at 60°C. After incubation the mixtures were diluted to 150 .walrnl -, RNA in the Sl nuclease buffer and divided in half. Half was treated with S 1 nuclease as described above, and the other half was treated similarly but without Sl nuclease. All incubations were to the same equivalent COfwith respect to DNA (2.14), to eliminate possible variability in data due to DNA renaturation. In order to determine the extent of DNA renaturation and the possible contamination of the RNA RNA preparations with DNA, RNA which had been hydrolyzed (0.3 N NaOH, 18 h at 37°C) was incubated with the DNA as above. Less than 1.1 % of the input radioactivity was S 1-nuclease-insensitive and this value was subtracted from the observed percents reassociated. Since this value was not greater than the value observed at that Cot in the excess of total
sperm DNA (fig. 2), the RNA preparations were not contaminated with DNA.
Thermal stability of hybrids Hybrids were diluted to 0.1 M NaCl and their thermal stability was determined by raising the temperature of the solution from 60 to 100°C in increments of 5°C. After equilibration for 5 min at each 5°C increment, an aliquot was removed and digested with Sl nuclease, and the S 1 nuclease-insensitive radioactivity was determined as above.
RESULTS Embryonic development
Animalized embryos develop into large thinwalled motile blastula-like structures covered with stereocilia and endowed with a small aggregate of cells in the blastocoel near the vegetal pole. Such embryos completely lack a gut at a stage when the gut of normal embryos is regionally differentiated (fig. 1). The viability of such embryos is equal to that of non-fed control embryos. Templates for rapidly-labelled heterogeneous RNA
Assayed by Sl nuclease the genome of the sea urchin appears to contain at least 30 % repeated sequences, and the non-repetitive component reassociates with a 8 Cot of Exptl Cell Res 92 (1975)
406
100'
' 0.01
Eckberg and Ozaki
I 0.1
I I
I IO
I 100
I 1000
I IO 000
Fig. 2. Abscissa: C,t; ordinate: % Sl nuclease insensitive. Reassociation kinetics of sea urchin suerm DNA assayed by Sl nuclease (0). Reassociation kinetics of radioactive non-repetitive gastrula DNA in the presence of a 1 000-fold excess of total sperm DNA assayed by S 1 nuclease ( l ).
during the development of normal and animalized embryos, RNA/DNA hybridization was carried out using purified radioactive non-repetitive DNA. That this fraction of DNA is essentially free of repetitive sequences is shown by its hybridization kinetics with total sperm DNA (fig. 2). The technique employed for saturation hybridization involved the incubation of labelled DNA of high specific activity with a large excess of non-radioactive RNA. At saturation, doubling of the RNA concentration or time of incubation results in no further increase in the per cent hybridization. A typical saturation curve is presented in fig. 4 and shows that, in the case of normal prism larvae, saturation is approached when 7.7 % of the DNA is hybridized. Assuming asymmetric transcription, this corresponds to approx. 15.4% of the non-repetitive DNA. The apparent saturation values obtained at all stagestested are listed in table 1. It should be pointed out that since saturation is never actually reached the data represent minimal estimates of the genomic information present. To compare the transcripts present at different stagesof the development of normal d
approx. 800 (fig. 2). In order to determine whether rapidly-labelled RNA is transcribed from repetitive or non-repetitive DNA sequencesand whether the relative transcription from repetitive and non-repetitive DNA sequences is different between normal and animalized embryos, RNA/DNA hybridization was carried out in DNA excess. RNAs from normal prism larvae and from animalized embryos of the sameageboth reassociated primarily with non-repetitive DNA (fig. 3). At a Cot of 30, 7-10s of the material was RNase-insensitive, but since 3-4s was RNase-insensitive at zero-time, the actual amount hybridized is probably on the order of 5 % at this Cot value. The 9 Cot for the reaction of the RNA with non-repetitive DNA is within a factor of 2 of that for the DNA reassociation. The scatter of the data precludes a more precise determination of the hybridization kinetics. Genomic representation in RNA of normal and animalized embryos
In order to assay directly for the amount of genomic information expressed in RNA Exptl Cell Res 92 (1975)
0, ,
I
I
I
I
I
I 0.1
I I
I IO
I 100
I 1000
I’
0 so
1 0.01
I 10000
Fig. 3. Abscissa: DNA C,,t; ordinate: % ribonucleaseinsensitive. Kinetics of hybridization of 30 mm pulse-labelled RNA from 0, normal prism larvae; 0, animalized embryos of the same age with a lOO-fold excess of total sperm DNA.
RNA in abnormal sea urchins
Table 1. Per cent of non-repetitive
DNA hybridized to RNA from various developmental stages of normal and animalized embryos
I” a-
I
0
IO
I
I
401
I a-
RNA pg/ml x h x 1O-4 Stage Unfertilized egg Blastula (24 h) Prism(48 h) Animalized (24 h) Animalized (48 h) Blastula + animalized (24 h)a Prism t animalized (48 h)’ Prism + blastulaa Animalized (24 h) + animalized (48 h)”
24 4.4 6.4 6.9 E 810 8.9 7.9
48 4.5 7.3 7.7 7.6 8.1
20
I
I
30
40
50
Fig. 4. Abscissa: RNA ,ug/ml x h x lo-$ ordinate: % DNA S 1 nuclease-insensitive. Saturation of radioactive non-repetitive DNA with RNA from 0, normal prism larvae; l , animalized embryos of the same age. RNA/DNA ratios ranged up to 10 000: 1. All incubations were to the same equivalent DNA Cot (2.14) to eliminate possible variation in results due to DNA renaturation.
7.1
a In additive experiments the concentration x time factor for each RNA reactant was 24 x lo* ,ug/ml x h.
and animalized embryos, additive experiments were performed in which the DNA was hybridized with RNAs from two different stages simultaneously. The difference between the arithmetic sum of the saturation values of each RNA preparation separately and that obtained in the additive experiments represents the extent of homology between the RNA populations. The results of these experiments are found in table 1. Since the concentration x time factor for each RNA reactant was 24 x lo4 pg/ml x h, the total concentration x time factor was twice that for RNA speciescommon to the two preparations. Thus an exact quantitative estimate of the homology between the populations cannot be made. However, by comparing the values from the additive experiment with those from the longer and shorter incubations, upper and lower limits can be placed on the extent of homology. For the prism + blastula experiment, 6.4 + 6.9-7.9 = 5.4, the lower limit of the per cent of the genome represented in both the normal blastula and prism. The upper limit is 7.3+7.7-7.9=7.1. Since (5.4/6.0)=0.79 and
(7.1/7.7) =0.92 we conclude that between 79 and 92% of the sequences present in the prism are also present in the blastula. By similar calculations, between 78 and 95 % of the non-repetitive DNA transcripts of 24 h blastulae are also present in 24 h animalized embryos; between 78 and 90% of the nonrepetitive DNA transcripts of the 48 h prism larva are also present in 48 h animalized embryos, and essentially 100% of the nonrepetitive transcripts in 24 h animalized embryos are retained in 48 h animalized embryos. Thus the difference between normal and animalized embryos of the same age is of the same magnitude as the difference between normal blastulae and prism larvae. Thermal stability of RNA/DNA
hybrids
The thermal stability of radioactive nonrepetitive DNA/total sperm DNA hybrids and that of radioactive non-repetitive DNA/ RNA hybrids is given in fig. 5. The DNA/ DNA hybrids melt very sharply with a T, of 78”C, whereas the RNA/DNA hybrids melt sharply with a T,,, of 71°C. The sharp melting profile, indicating that all hybrids are of equal stability, is characteristic of non-repetitive RNA/DNA hybrids and not of repetitive RNA/DNA hybrids. The observed depression is due, at least in part, to Exptl Cell Res 92 (1975)
408
Eckberg and Ozaki
60
40
20
0 6
Fig. 5. Abscissa: temp. (“C); ordinate: % S 1 nucleaseinsensitive radioactivity released. Thermal denaturation profiles of radioactive nonrepetitive DNA/total sperm DNA hybrids (0); DNA/RNA hybrids (0), in 0.1 M Na+, assayed by Sl nuclease. T, of DNA/DNA hybrids is 78°C; T,,, of RNA/DNA hybrids is 71°C.
short hybrid regions [15, 331. The maximum length of the hybrids is about 200 nucleotide pairs as limited by the size of the RNA molecules hybridized. With the above considerations and the fact that well-matched bacterial RNA/DNA hybrids can melt with approximately a 5°C drop in T,,, [4], we conclude that very little mismatch is present, and thus that near locus specificity has been achieved. DISCUSSION RNA transcripts have been studied in animalized and normal sea urchin development by homology with various regions of the genome by RNA/DNA hybridization. Sucrose gradient analysis demonstrates that most of the radioactivity incorporated by prism larvae and by animalized embryos ExpfI Cell Res 92 (1973)
during a short pulse label is incorporated into heterogeneously sedimenting high molecular weight material, most likely heterogeneous nuclear RNA (hnRNA) [12]. In order to determine whether this material is synthesized from repetitive or non-repetitive DNA sequences, this material was hybridized to DNA in an excessof DNA. When RNA is present in trace quantities in a DNA/DNA reassociation mixture, the RNA will hybridize to the DNA and the kinetics of hybrid formation will reflect the degree of reiteration of the homologous DNA in the genome. For the interpretation of such experiments, the DNA excess must be large; at least 70-loo-fold [3]. In the experiments reported here, the DNA excess was loo-fold, but it should be mentioned that less than 10% of the cellular RNA in the reaction mixture is the hnRNA in which nearly all the label is found. Thus it is likely that the DNA is in at least a 1000-fold excess over hybridizable radioactive RNA sequences. Under these conditions, the reaction should give an accurate picture of the reiteration frequency of the genes transcribing hnRNA in normal and animalized seaurchin embryos. From these data we conclude that the representation in hnRNA of the repetitive sequences of the sea urchin genome is small. Smith et al. [33], using purified gastrula hnRNA and a DNA excessof over 100 OOOfold obtained similar results. The nature of the zero-time RNase-insensitive material was not further studied, but this fraction may correspond to the fraction of hnRNA known to be double-stranded [18]. This material could be rendered RNase-insensitive either through intramolecular base pairing or through its rapid hybridization to DNA 1171. In order to assay for the complexity of RNA transcripts present at different stages of development, RNA/DNA hybridization
RNA in abnormal sea urchins
was carried out using purified non-repetitive DNA in a large excess of RNA. Under such conditions, the contribution of the DNA to the rate of hybridization may be ignored and the reaction is pseudo firstorder. In experiments using total cellular RNA the actual concentration of hybridizable RNA sequencesis unknown since most of the cellular RNA is ribosomal (rRNA) and transfer RNA (tRNA) which do not bind to non-repetitive DNA [7, 25, 341. Nevertheless, saturation plateaus can be determined and the extent of homology between two populations of RNA can be estimated. Although purified non-repetitive DNA fractions will contain, in the limit, one copy of each repetitive DNA sequence, all or nearly all the observed hybridization must be to non-repetitive sequences, since this DNA is vastly enriched for non-repetitive sequences, and nearly all rapidly-labelled heterogeneous RNA ([33] and fig. 3) and all messenger RNA [14] hybridize to nonrepetitive DNA. The data of table 1 demonstrate that during the development of both normal and animalized embryos the complexity of transcription increases and that stage-specific patterns are observed. Transcripts are present at each stage tested which are unique to that particular stage. It is of particular interest that relatively large differences are observed between the unique DNA sequence transcripts of normal and animalized embryos of the same age. The data of table 1 also demonstrate that the homology between the RNAs derived from non-repetitive DNA of different stages of development is similar to that of RNAs derived from repetitive DNA sequences[38]. In addition it may be noted that the saturation levels obtained at different stages of development correspond roughly to the complexity of transcription at analogous
409
stagesin the development of other organisms. Davidson & Hough [lo] saturated 0.9 % of the non-repetitive portion of the Xenopus laevis genome with unfertilized egg RNA. Since the Xenopus genome is about 4 times as large as that of the sea urchin, the complexity of the stored RNA messagesin the unfertilized Xenopus egg is similar to that in the unfertilized sea urchin egg. Davis & Wilt [ll] have reported that the unfertilized egg of the marine worm, Urechis caupo, which has a genome slightly larger than that of the sea urchin, contains RNA complementary to 4.3 % of its non-repetitive DNA. Thus the RNA complexity in the unfertilized egg of Urechis is nearly the same as that in the unfertilized sea urchin egg. Schultz et al. [32] have shown that 1.8 % of the nonrepetitive portion of the rabbit genome is represented in RNA in preimplantation blastocysts and that 2.5 % is represented in post-implantation blastocysts which have begun to differentiate. Since the rabbit genome is about 4 times as large as that of the seaurchin, the actual amount of information in these stagesof rabbit embryos is very close to that present in sea urchin embryos at the analogous stages. Similar values have been reported for early mouse development [9]. Such results are not surprising as the embryos of all these organisms are carrying out similar activities at analogous stages;e.g. mitosis, primary germ layer formation, maintenance of metabolism, etc. The high degree of homology between the RNAs of different stagesof development may reflect great stability of messages.The unfertilized egg contains messagessufficient to support development through the blastula stage [35]. It is possible that some of these messagesare retained for long periods during development. It is believed that during development messagesare transcribed long before they are to be used by the embryo to support Exptl Cell Res 92 (1975)
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differentiation [13, 35, 381. The presence of such long-lived messageswould lead to the observed high degree of homology between the RNAs present at different stages of development. The half-life of newly-synthesized mRNA in sea urchin embryos has been reported to be about 75 min [5]. Recent measurementshave indicated that in cultured cells the half-life of mRNA is much longer, however [24]. The existence of even a small fraction of very long-lived mRNA in sea urchin embryos might provide the results obtained. Alternatively, most of the messages in RNA in the early sea urchin embryo may not be concerned with differentiation, but rather with activities common to all cells. It has long been known that sea urchin development can be altered by various operative and chemical methods. Various types of experiments have implicated the genome in the control of sea urchin development and its experimental alteration. Treatment with actinomycin D reduced the animalization of isolated animal half embryos [22] and that of whole embryos chemically animalized [19, 211.Animalization also alters the protein synthetic pattern. Esterase isozyme activities which appear in normal pluteus larvae do not appear in animalized embryos of the same age [26], and there are differences between the newly-synthesized proteins of normal and animalized embryos [8]. The present results demonstrate that alterations in the pattern of embryonic gene expression do occur in animalization, and therefore that these alterations can be conveniently induced in embryos merely by altering their ionic environment. Taken together with the results cited above, they suggest that alterations in genetic activity may, indeed, lead to the developmental abnormality known as animalization. This research was supported in part by a Grant-in-Aid of research from the Society of the Sigma Xi to ExptI Cell Res 92 (1975)
W. R. E. and by a Bio-Medical Sciences Support grant from Michigan State University to H. 0.
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2. Bellamy, A R & Ralph, R K, Meth enzymol 12B (1968) 156. 3. Bishou. J 0. Biochem i 126 (1972) 171. 4. Bolton; E T & McCarthy,‘ B J, J mol biol 8 (1964) 201. 5. Brandhorst, B P & Humphreys, T, J cell biol 53 (1972) 474. 6. Britten, R J & Kohne, D E, Science 161 (1968) 529. 7. Brown, D D & Weber, C S, J mol biol 34 (1968) 661. 8. Carroll, A G, Eckberg, W R & Ozaki, H, Exptl cell res 90 (1975) 328. 9. Church, R B & Brown, I R, Nucleic acid hybridization in the study of cell differentiation (ed H Ursprung) p. 11. Springer, New York (1972). 10. Davidson; E H & Hough, B R, J mol biol 56 (1971) 491. 11. Davis, F C &Wilt, F H, J cell biol 59 (1973) 72a. 12. Eckberg, W R & Ozaki, H, Exptl cell res 73 (1972) 177. 13. Giudice, G, Mutolo, V & Donatuti, G, Wilhelm Roux’ Arch Entwicklungsmech Organ 161 (1968) 118. 14. Goldberg, R B, Galau, G A, Britten, R J & Davidson, E H, Proc natl acad sci US 70 (1973) 3516. 15. Hayes, F N, Lilly, E H, Ratliff, R L, Smith, D A & Williams, D L, Biopolymers 9 (1970) 1105. 16. Horstadius, S, Biol rev 14 (1939) 132. 17. Jelinek, W & Darnell, J E, Proc natl acad sci US 69 (1972) 2537. 18. Kronenberg, L H & Humphreys, T, Biochemistry 11 (1972) 2020. Lallier, R, Compt rend acad sci 257 (1963) 2159. :;: Leong, J-A, Garapin, A-C, Jackson, N, Fanshier, L, Levinson, W & Bishop, J M, J viral 9 (1972) 891. 21. Markman. B & Runnstrom. J. Exotf cell res 31 (1963) 615. 22. - Wilhelm Roux’ Arch Entwickhmgsmech Organ 165 (1970) 1. 23. Mizuno, S, Whiteley, H R & Whiteley, A H, Differentiation 1 (1973) 339. 24. Murphy, W & Attardi, G, Proc natl acad sci US 70 (1973) 115. 25. Mutolo, V & Giudice, G, Biochim biophys acta 149 (1967) 291. 26. O’Melia, A F, Exptl cell res 73 (1972) 469. 27. O’Melia A F & Villee. C A. Exntl cell res 72 (1972) 276. 28. Pirrone, A M, Sconzo, G, Mutolo, V & Giudice, G, Wilhelm Roux’ Arch Entwicklungsmech Organ 164 (1970) 222. 29. Runnstrom, J, Wilhelm Roux’ Arch Entwicklungmech Organ 129 (1933) 442. z,
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35. Tyler, .A, Dev biol, suppl. 1 (1967) 170. 36. Vogt, V M, Eur j biochem 33 (1973) 192. 37. Wetmur, J G & Davidson, N, J mol biol 31 (1968) 349. 38. Whiteley, H R, McCarthy, B J & Whiteley, A H, Dev biol 21 (1970) 216. Received October 22, 1974 Revised version received December 2, 1974
Exptl Cell Res 92 (1975)