Synthesis of heterogeneous mRNA-like RNA and low-molecular-weight RNA before the midblastula transition in embryos of Xenopus laevis

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BIOLOGY

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(1987)

Synthesis of Heterogeneous mRNA-like RNA and Low-Molecular-Weight RNA before the Midblastula Transition in Embryos of Xenopus Levis NORIHIKO NAKAKURA, TAKAYO MIURA, K. YAMANA, AKIO ITO,* AND KOICHIRO SHIOKAWA Laboratory

of Developmental Biology, *Laboratory of Biochemistry, Department Faculty of Science 33, Kyushu University, Fukuoka, 812 Japan Received December 8, 1986; accepted in revised

form May

of Biology,

6, 1987

It has been proposed and is now widely accepted that in Xenom laewis embryogenesis RNA synthesis starts only at and after 12 rounds of cleavage, at the time of the midblastula transition (MBT). In this report, however, we provide evidence that RNA synthesis takes place prior to the MBT stage in normally developing Xenqpus embryos. In the present experiments, we cultured fertilized eggs in 80 mM phosphate buffer and loosened the adhesion between blastomeres, so that rH]uridine could be incorporated into blastomeres from the surrounding medium. By this method and also by microinjection of rH]GTP, we found that embryos synthesize heterogeneous, nonribosomal, high-molecular-weight RNAs and a relatively small amount of low-molecular-weight RNA as early as the sixth cleavage. RNAs synthesized were not of mitochondrial origin, and the synthesis was sensitive to actinomycin D and a-amanitin. From these results we conclude that mRNA-like RNA and low-molecular-weight RNA start to be synthesized during the cleavage stage. 0 1987 Academic

Press, Inc.

age, while keeping the vitelline membrane intact (Takeichi et al, 1985; Shiokawa et al, 1985), and we labeled embryos by incubating them in a very high concentration (0.4-0.5 mCi/ml) of rH]uridine added into the surrounding medium. The results obtained clearly showed that heterogeneous mRNA-like RNA as well as a small amount of low-molecular-weight RNA is synthesized during cleavage prior to the MBT stage in the normal embryogenesis of Xenopus Levis.

INTRODUCTION

In Xenopus laevis, fertilized eggs undergo 12 rounds of synchronous division and reach the stage that is called midblastula transition (MBT) (Signoret and Lefresne, 1971; Gerhart, 1980;Newport and Kirschner, 1982a). New gene expression from the zygotic nuclei of the fertilized eggs has been proposed to commence at the MBT (Newport and Kirschner, 1982a,b). To show this, Newport and Kirschner (1982a) injected fertilized eggs with [32P]rUTP and reported that the syntheses of 4 S RNA, Ul and U2 snRNAs, 5 S RNA, 7 S RNA, and mRNA take place roughly at the MBT, but not before the MBT stage. In our previous studies (Shiokawa et aZ., 1981a,b), however, we labeled dissociated cells obtained from demembranated Xenopus cleavage stage embryos with [methyl-3H]methionine and found that methylation of the 5’-cap structure of mRNA occurs already at the cleavage stage (at around stages 6-8). In an in vitro system with HeLa cell extract, the formation of the Y-cap structure on mRNA has been reported to be coupled with transcription initiation (Manley et aL, 1979; Jove and Manley, 1982). Therefore, it was quite tempting to assume that the methylation of cap structure found during stages 6-8 (Shiokawa et al, 1981a,b) reflects the transcription of mRNA before the MBT stage. In the present experiment we paid attention mainly to the possibility of the occurrence of RNA synthesis during the cleavage stage. For this purpose, we made use of 80 mM phosphate buffer to loosen the tight connection of blastomeres from the beginning of the cleav-

MATERIALS

AND

METHODS

Labeling of Embryos Embryos of Xenopua laevti were obtained by artificial fertilization (Tashiro et ak, 1983) and cultured at 2122’C throughout the experiment. Embryos were dejellied and immediately immersed in 80 mM phosphate buffer (PB) (70 mM Na2HP04, 10 mM NaH2P04, pH 7.6) containing 100 pg/ml of penicillin and 50 pg/ml of streptomycin. Cellular connection started to loosen soon after the initiation of cleavage (Takeichi et al, 1985; Shiokawa et al., 1985; Atsuchi et aZ.,1986). Figure 1 shows cleaving embryos cultured in PB. The stages of the embryos shown are fifth (Fig. lA), sixth (Fig. lB), and seventh (Fig. 1C) cleavage, as determined from a comparison of these with the cleavage patterns of sibling whole embryos which were followed by time-lapse cinematography. It is apparent that blastomeres were separated from each other within the vitelline membrane, keeping their spatial orientation relatively unchanged (Takeichi et al.,

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0 1987 by Academic Press, Inc. of reproduction in any form reserved.

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then cultured either in 80 mM phosphate buffer or in doubly modified amphibian Ringer solution (MMR) (0.1 M NaCl, 2 mM KCl, 1 mM MgSO,, 2 mM CaClz, 5 mM Hepes, 0.1 mM EDTA, pH 7.8) (Ubbels et all, 1983; Newport and Kirschner, 1982a) as a nondissociating medium, both containing penicillin and streptomycin as above. The labeled embryos were frozen in liquid nitrogen and stored at -20°C until analyzed. Unless otherwise stated, 30 embryos were used to obtain an RNA sample. RNA Extraction and Fractionation Frozen embryos were homogenized in 0.1 M sodium acetate buffer (pH 5.0) containing 10 pg/ml of bentonite and 0.5% SDS (sodium dodecyl sulfate) (Shiokawa and Yamana, 1967). Homogenates were mixed with phenol and then vigorously shaken at 20-25°C for l-2 hr in a gyratory shaker (Shiokawa et aL, 1986a). RNA was precipitated from the aqueous phase with 0.2 M NaCl and 2.5 vol of ethanol. RNAs were electrophoresed on lo-cm gels of 0.5% agarose-2.4% polyacrylamide as described previously (Shiokawa et aL, 1979). Gels were scanned in a densitometer at 260 nm to locate the peaks of 18 S and 28 S rRNAs and 4 S RNA on the electrophoretic profiles of RNAs. Efficiency of the extraction of the labeled RNA was over 80%) as determined by the recovery of uv-absorbing materials (one embryo contains cit. 4000 ng RNA) (Shiokawa and Yamana, 1967). Cell Fractionation for Preparation of Mitochondrial and Other Fractions

FIG. 1. Appearance of embryos cultured in PB. Fertilized eggs were immersed in PB at 200 embryos/4 ml and embryos were “dissociated” within the vitelline membrane. Embryos were filmed at 3.5 hr (A), 4 hr (B), and 4.5 hr (C) after fertilization. The corresponding whole embryos were cultured in MMR, and cleavage of these embryos was followed by time-lapse cinematography. The stages of the whole embryos corresponding to (A), (B), and (C) were fifth, sixth, and seventh cleavage stages, respectively.

1985; Shiokawa et al., 1985; Atsuchi et al., 1986). Blastomeres of these embryos continued to divide apparently normally as in the control whole embryos at least until the gastrula stage (Atsuchi et aL, 1986). These embryos were labeled by adding rH]uridine-5T (25 Ci/mmole) into the surrounding medium at 400-500 &i/ml. In one of the experiments, fertilized eggs were microinjected with [3H]rGTP (10 Ci/mmole) according to the methods described previously (Shiokawa et al,, 1983; 1986b; 1987; Tashiro et aL, 1986). The injected eggs were

Twenty embryos labeled with [3H]uridine were homogenized in 0.25 M sucrose containing 0.003 M TrisHCl (pH 7.4), 0.001 M EDTA, and 50 pg/ml heparin in a Dounce-type homogenizer. The homogenate was fractionated by differential centrifugation into nuclear (500 rpm, 10 min, pellet), mitochondrial(l2,OOO rpm, 15 min, pellet) and cytoplasmic (12,000rpm, 15 min, supernatant) fractions. It was confirmed that the mitochondrial fraction contains practically the total cellular activity of cytochrome oxidase, an indicator enzyme of mitochondria as assayed by methods described previously (Boell and Weber, 1955; Wharton and Tzagoloff, 1967). RESULTS

Pattern of RNA Labeling in Pre-MBT Embryos Embryos cultured in PB were administered with [3H]uridine at 1.5 hr after fertilization. When embryos were labeled for 2.75 hr until 4.25 hr of fertilization, heterogeneous radioactivity peaks appeared in the highmolecular-weight RNA region, but not in the 4 S RNA

NAKAKURA ET AL.

RNA

Synthesis

region (Fig. 2A). The main peak was located in the middle of the peaks of 18 S and 28 S rRNAs. Therefore, it appears that the labeled high-molecular-weight RNA was not ribosomal. The labeling obtained here was assumed to be due to RNA synthesis, since the label disappeared completely from the high-molecular-weight RNA region when the RNA preparation was incubated for 30 min with RNase (100 pg/ml) prior to electrophoresis (profiles omitted). When labeling lasted longer, the amount of labeled high-molecular-weight RNA increased, and a relatively small and broad peak also appeared around the 4 S RNA region. Figure 2B shows such a profile for RNA labeled for 4.2 hr until 5.75 hr after fertilization. These labeling patterns contrasted sharply with those obtained from embryos after the MBT stage, in which the labeling of 4 S RNA was extremely high (see below and also Shiokawa et al., 1979). When labeled for 4.75 hr until 6.25 hr after fertilization, there appeared another peak in the high-molecular-weight RNA region that migrated slightly slower than 28 S rRNA. Figure 2C shows such a peak of high-molecularweight RNA labeled for 5.25 hr until 6.75 hr after fertilization. The incorporation increased in all the RNA components throughout the gel, but the general feature of the profile was the same as those obtained with shorter labeling, except that the peak at approximately 4 S RNA became relatively larger. A similar profile was obtained even when labeling lasted for 5.75 hr until 7.25 hr after fertilization, when embryos had almost reached the midblastula stage (MBT) (profile not shown).

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FIG. 2. Patterns of RNAs labeled in embryos during the pre-MBT stage. Fertilized eggs were immersed in PB at 206 embryos/l ml, and embryos were administered FHjuridine at 400 &i/ml at 1.5 hr after fertilization. Fifteen embryos each were withdrawn at 4.25 hr (A), 5.75 hr (B), and 6.75 hr (C) after fertilization and immediately frozen in liquid nitrogen. RNAs were extracted from the labeled embryos and electrophoresed on gels. The approximate locations of 4 S RNA and 18 S and 28 S rRNAs are indicated in these and the following profiles.

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FIG. 3. Kinetic change in the amounts of labeled high-molecularweight and low-molecular-weight RNAs during the cumulative labeling in the experiment in Fig. 2. The amounts of labeled RNAs were calculated, based on the profiles obtained, as in Fig. 2. F, fertilization; PB and rH]UR, addition of PB and [3H]uridine, respectively. Highmolecular-weight RNA (0), low-molecular-weight RNA (0).

The Earliest

Stage of Labeling

of RNA

To determine the earliest stage at which RNA synthesis was detectable, we repeated the above experiment and followed the cleavage cycles accurately. For this purpose, we used time-lapse cinematography. When we added [3H]uridine to the PB-immersed embryos at 2 hr after fertilization and labeled them for 1.5 hr, embryos reached the fifth cleavage (cf. Fig. lA, for the appearance of embryos). At this time point, we could not detect a significant incorporation of [3H]uridine throughout the gel (data not shown). However, 30 min after this, when embryos were at the sixth cleavage (cf. Fig. lB), we succeeded in obtaining a detectable labeling of heterogeneous RNA. The profile obtained was essentially the same as that in Fig. 2A and is not reproduced here. When we labeled the embryos for 2.5 hr (until the seventh cleavage; cf. Fig. lC), the incorporation increased, but the profile was still as in Fig. 2A. Therefore, the earliest stage at which we successfully detected the synthesis of high-molecular-weight RNA was the sixth cleavage under the present conditions. Changes in the Relative Amounts of RNAs Accumulated during Cleavage Stage

Based on the results of the experiments described above, we calculated the amounts of label accumulated in high-molecular-weight RNA and also the low-molecular-weight RNA and plotted these values as a function of the time after fertilization (Fig. 3). In Fig. 3, which covers the time interval from fertilization to MBT, it is quite apparent that embryos synthesize and accumulate heterogeneous high-molecular-weight RNA and a rela-

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tively smaller amount of small-molecular-weight RNA with a rate twice as great as that for high-molecularin the latter half of the period. weight RNA. Extrapolation of the curve for the lowFrom the data of Fig. 3, it appears that the amount molecular-weight RNA suggests that the synthesis of of label accumulated in RNA almost doubles every 35 this RNA starts at about 4 hr after fertilization, and by min. We assume that RNA synthesis proceeds simply the time of the MBT stage, its amount exceeds that of in proportion to the number of nuclei, which increases the high-molecular-weight RNA. Therefore, the data in approximately exponentially with the 35-min cell cycle Fig. 4 suggest that while the rate of the synthesis of (Satoh, 1977; Suzuki et al., 1976; Shiokawa and Yamana, high-molecular-weight RNA per nucleus was approxi1979). mately constant, that of low-molecular-weight RNA inTo make this point clearer, we replotted the data in creases with time to become larger than that of highFig. 3, taking the logarithms of the values of incorpo- molecular-weight RNA at and after the MBT stage. rated label. In Figure 4, it is apparent that the increase of accumulated label in both high-molecular-weight Evidence That the Pre-MBT Synthesis Is Not Due to a RNA and low-molecular-weight RNA was linear in this Disturbance of the Cell Cycle semilogarithmic presentation. In fact, the amount was almost doubled every 35 min in the high-molecularIn Drosophila embryos, it was recently reported that weight RNA, Therefore, it appears that the rate of syn- cycloheximide, an agent that elongates the cell cycle, thesis per nucleus of high-molecular-weight RNA is not causes premature gene expression by zygotic nuclei (Edincreasing but is constant at least during the pre-MBT gar and Schubiger, 1986). Since the data obtained above stage examined. Furthermore, the extrapolation of the were all derived from the labeling of Xenopus embryos curve of the high-molecular-weight to the zero time of dissociated in PB, it is possible that the dissociation in fertilization strongly suggests that there is synthesis of PB may have caused a disturbance in the cell cycle, which high-molecular-weight RNA even at the very beginning secondarily caused precocious activation of RNA synof cleavage, although we could not detect the synthesis thesis in a way which does not occur in normal undis(see above). sociated embryos. By contrast, the increase in the amount of label in To test this possibility, we injected 60 uncleaved ferlow-molecular-weight RNA was much steeper than that tilized eggs with [3H]GTP and transfered 30 each into in high-molecular-weight RNA. Thus, for low-molecularMMR and PB. The embryos were then cultured for 4.5 weight RNA the amount of incorporated label increased hr (during the pre-MBT stage) or 8 hr (until the MBT stage). The labeling patterns under the two different culture conditions were then compared and found to be quite similar to one another, irrespective of whether labeling was early in the pre-MBT (Figs. 5A and 5B) or until the MBT stage (Figs. 5C and 5D). Therefore, we conclude that the pre-MBT RNA synthesis detected above in PB-dissociated embryos is not an artifact due to the disturbance of the cell cycle or to other unknown factors that may be caused by the dissociation of embryos in PB. Comparison of the Pattern of RNA Synthesis before and after the MBT Stage

Time after fertilization

(hrl

FIG. 4. Semilogarithmic. presentation of the accumulation of the label in high-molecular-weight RNA and low-molecular-weight RNA. Data were transferred from the results in Fig. 3. High-molecular-weight RNA (0), low-molecular-weight RNA (0).

Embryos dissociated in PB were labeled for 3 hr starting from the 3 hr after fertilization until stage 7.5 (labeling during the pre-MBT stage) and also for 6.5 hr until stage 9 (labeling until shortly after the MBT stage). When these two RNA profiles were compared, it was apparent that while the labeling of low-molecularweight RNA was relatively much smaller in the preMBT stage (Fig. 6A), it was greatly enhanced shortly after the MBT stage (Fig. 6B). On the ordinate of the two profiles there is approximately a loo-fold difference.

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in Xenopus

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synthesis of RNA, especially of low-molecular-weight RNA, at the time of the MBT. Eflects of Actinomycin D on Pre-MBT RNA Synthesis To study the nature of the pre-MBT RNA synthesis, we examined the effects of actinomycin D by incubating dissociated embryos in PB that contained the drug. The dose of the drug was selected to be 10 pg/ml, since the labeling duration was relatively short and since the blastomeres were extremely large in size and appeared to be less sensitive than cells of later stage embryos. Embryos in PB were exposed to actinomycin D (10 pg/ ml) at 1.5 hr after fertilization, and 1 hr later they were supplemented with [3H]uridine and labeled for 4 or 5 hr in the continued presence of actinomycin D. In the cul100 50 75 0 25 100 50 75 0 25 tures labeled for 4 hr (labeling during the pre-MBT Distance moved (mm) stage), it was found that about 75% of the labeling of FIG. 5. Comparison of the patterns of RNA synthesis between whole high-molecular-weight RNA and about 85% of low-moembryos and dissociated embryos. Fertilized eggs were microinjected lecular-weight RNA was inhibited (Fig. 7B). In the lawith 30 nl of [SH]GTP (5 &i/rl). Embryos were immersed either in beling for 5 hr (labeling until at the MBT stage), about MMR (A, C) or in PB (B, D) at 0.5 hr after the fertilization. Twenty 85% of the labeling of high-molecular-weight RNA was embryos were withdrawn after the labeling for either 4.5 hr (before the MBT stage) (A, B) or 8 hr (until the MBT stage) (C, D). RNAs inhibited, although in the labeling of low-molecularwere extracted from the labeled embryos and fractionated as in Fig. 2. weight RNA, the inhibition was less (around 60%; Fig. 7D). These results show that most of the labeling of both high-molecular-weight and low-molecular-weight RNA Assuming that the cell number differs approximately was due to DNA-dependent RNA synthesis. The relalo-fold between the two stages, there appears to be tively larger resistance of the labeling of low-molecularroughly a lo-fold increase per nucleus in the accumu- weight RNA in the longer labeling period may suggest lation of label before and after the MBT stage. Assuming that this is the case, the activation of the synthesis of low-molecular-weight RNA would be about loo-fold, 1 A since the peak height of the low-molecular-weight RNA 26316s in the post-MBT embryos was about 10 times that obII ‘S 4tained for pre-MBT stage embryos. Thus, it appears that there is a large increase (lo- to loo-fold) in the rate of

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FIG. 6. Comparison of the patterns of RNA synthesis before and after the MBT stage. Embryos were dissociated in PB and labeled with [sH]uridine (500 &i/ml) either for 3 hr after the third hr of fertilization to stage 7.5 (A) or for 6.5 hr to stage 9 (B). RNAs were extracted from the labeled embryos and fractionated as in Fig. 2.

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FIG. ‘7. Effects of actinomycin D treatment on the pattern of RNAs labeled during cleavage stage. Fertilized embryos were immersed in PB and at 1.5 hr after fertilization they were administered actinomycin D at 10 pg/ml. Embryos were incubated for 1 hr in the actinomycin D-containing medium and then further administered [BH]uridine at 500 &i/ml. Fifteen embryos were withdrawn after labeling for either 4 hr (A, B) or 5 hr (C, D). RNAs were extracted and fractionated as in Fig. 2. (A and C) Control, (B and D) actinomycin D-treated.

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the occurrence of some CCA-terminal exchange in 4 S RNA, since this type of exchange is resistant to actinomycin D (Franklin, 1963) and is known to occur in early Xenopus embryos (Brown and Littna, 1966). Eflects of a-Amanitin on Pre-MBT RNA Synthesis Embryos in PB were exposed to a-amanitin (100 pg/ ml) at 1.5 hr after fertilization, and 1 hr later they were supplemented with [3H]uridine and labeled for 3.5 hr (during the pre-MBT stage; Fig. 8B) or 5.5 hr (until at the MBT stage; Fig. 8D). The inhibition in the labeling of high-molecular-weight RNAs was almost complete for both labeling periods, while the inhibition of lowmolecular-weight RNA synthesis was relatively smaller. It has been reported that a-amanitin at low doses specifically inhibits nucleoplasmic RNA polymerase II (Tata et al., 1972;Lindell et al., 1970) and, at higher doses, RNA 25 50 75 polymerases II and III (Weinmann and Roeder, 1974), Distance moved (mm) but not RNA polymerase I (Hastie and Mahy, 1973). FIG. 9. Distribution of RNAs in three cellular fractions. Twenty However, in this type of in wiwo experiment it is difficult to know to what extent the drug acted differentially on fertilized eggs immersed in 0.5 ml of PB were administered 400 j&i/ each of the RNA polymerases. (For instance, a low dose ml of [aH]uridine 1.5 hr after fertilization and labeled for 7 hr until stage 8.5 (MBT stage). Embryos were fractionated into the cytoplasmic of a-amanitin at 1 pg/ml was found to be ineffective in (A), mitochondrial (B), and nuclear (C) fractions. RNAs were extracted the preliminary experiment.) Nevertheless, the results and fractionated as in Fig. 2. provide evidence that the majority of the high-molecular-weight RNA synthesized during the cleavage stage may be the product of RNA polymerase II, since if it were rRNA its synthesis would be resistant even to the high dose of cy-amanitin used. * ~l8.s

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FIG. 8. Effects of a-amanitin treatment on the pattern of RNA synthesis in cleavage stage embryos. Fertilized eggs cultured in PB for 1.5 hr were administered a-amanitin at 100 *g/ml. After being incubated for 1 hr, embryos were further administered [‘Hjuridine at 400 &i/ml. The labeling continued either for 3.5 hr (A, B) or 5.5 hr (C, D). RNAs were extracted and fractionated as in Fig. 2. (A and C) Control, (B and D) a-amanitin-treated.

Origin of the Labeled RNAs

The sensitivity of the labeling of high-molecularweight RNA to a-amanitin in Fig. 8 suggests the nonmitochondrial nature of the labeling, since mitochondrial RNA synthesis is known to be resistant to CYamanitin (Merten and Pardue, 1981). However, early Xenopus embryos contain an extremely large amount of mitochondria (Dawid, 1966; Callan et al, 1980; Chase and Dawid, 1972) which may maintain some RNA synthetic activity throughout development (cf. Chase and Dawid, 1972; Young and Zimmerman, 1973). Therefore, we fractionated the contents of labeled embryos to test whether the synthesis of RNA in pre-MBT embryos is due to mitochondria. For this purpose, we labeled embryos with [3H]uridine from 1.5 to 7.5 hr after fertilization (until the MBT stage) and examined the distribution of labeled RNA in the cytoplasmic, mitochondrial, and nuclear fractions. The results showed that about 80% of both high-molecularweight and low-molecular-weight RNAs exist in the cytoplasmic fraction, with the remaining 20% in the nuclear fraction, but none in the mitochondrial fraction (Fig. 9). Therefore, it is clear here that pre-MBT RNA

NAKAKURA

ET AL.

RNA Synthesis in

synthesis is not due to mitochondrial RNA synthesis. The recovery of the majority of the label in the cytoplasmic fraction may be due to an artifact such as nuclear breakdown during fractionation. However, these results were highly reproducible. At present, we prefer the interpretation that nuclear transcripts are quickly dispersed into the cytoplasm, since embryonic nuclear membranes breakdown frequently during the rapid cleavages. DISCUSSION

It is now widely accepted that synthesis of all the major classes of RNAs starts only at and after the MBT stage (Newport and Kirschner, 1982a). In fact, the first activation of rDNA expression was defined by ourselves to be at or shortly after the MBT stage as shown by detection of rRNA-specific 2’-0-methylation (Shiokawa et al, 1981a,b) and by Busby and Reeder (1983) as shown by S-l protection of the transcript from injected rDNA clones. Furthermore, the concept of MBT-associated activation of RNA synthesis has been strengthened by recent reports on the expression of exogenously introduced DNAs in the developing embryos. Thus, MBT-associated activation has been reported for the expression of the genes for prokaryotic chloramphenicol acetyltransferase (Etkin and Balcells, 1985; Fu and Shiokawa et al., unpublished observation), Drosophila alcohol dehydrogenase (Etkin et al, 1984), sea urchin histone (Bendig, 1981), rabbit P-globin (Rusconi and Schaffner, 1981), yeast tRNA (Newport and Kirschner, 1982b), and adult Xenopus LY-and /3-globin (Bendig and Williams, 1983). In spite of the concept put forth by Newport and Kirschner (1982a), however, we had an impression that some mRNA synthesis occurs even prior to the MBT stage, since we had previously found the capping of mRNA during the pre-MBT stage (stages 6 to 8) (Shiokawa et al., 1981a,b). Consist with the expectation, we now have clearly shown that embryos at the pre-MBT stage synthesize high-molecular-weight, nonribosomal RNAs and, in addition, a relatively smaller amount of low-molecular-weight RNA. In Drosophila, it has been reported that a critical stage similar to the Xenop MBT occurs after a certain number of cell divisions, and that the activation of RNA synthesis is also related to this stage (Edgar and Schubiger, 1986). Furthermore, it was demonstrated that RNA synthesis can be activated significantly earlier than normal if the interphase period of the embryonic cell cycle is prolonged by treatment of the cells with cycloheximide (Edgar and Schubiger, 1986). In our case with Xenopus laevis embryos, we did not use any drug to modify the cell cycle, and yet we found nonmitochondrial RNA synthesis in embryos during the

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pre-MBT stage. Therefore, it should be pointed out that our results are different from those of Edgar and Schubiger (1986) in that ours suggest the occurrence of preMBT RNA synthesis as a normal course of events. By contrast, what Edgar and Schubiger (1986) demonstrated was that cleavage embryos could be forced to synthesize RNA by being artificially modified in their cell cycles. Like those in Drosophila, cell cycles in Xenopus embryos are also characterized by the absence of G1 and Gz phases (Newport and Kirschner, 1982a). Therefore, it is surprising that RNA synthesis occurs during such unusually rapid cell cycles, since it has been reported that RNA synthesis stops almost completely during mitosis (Prescott and Bender, 1962; Konrad, 1963). However, in human cultured cells RNA synthesis can occur in S phase, and it continues on until early prophase (Prescott and Bender, 1962) or even until late prophase (Feinendegen and Bond, 1963). The continued synthesis of poly(A)+ RNA during S phase has also been reported in Chinese hamster ovary cells (Nobis et al, 1978). Furthermore, in HeLa cells, [14C]uridine is incorporated during S phase not only into high-molecular-weight RNA but also into low-molecular-weight RNA around the 4 S RNA region (Scharff and Robbins, 1965). Therefore, it may not be unreasonable to assume that the syntheses of mRNA-like high-molecular-weight RNA and low-molecular-weight RNA can take place during the Xenopus cleavage stage, despite the rapid cell division cycles. In the present experiments, the earliest stage at which we detected RNA synthesis was the sixth cleavage. At this stage, only high-molecular-weight RNA, but not low-molecular-weight RNA, was found to be synthesized. We do not know if our failure to detect RNA synthesis at fifth cleavage was due to the absence of RNA synthesis or to the insensitivity of the methods. However, if we accept the finding of RNA synthesis at the sixth cleavage in Xenopus embryonic cells, we ought to consider that RNA is synthesized at the same rate per nucleus (except for rRNA) even at the stages earlier than the sixth cleavage. The extrapolation of a semilogarithmic plot of the label accumulation curve strongly supports this prediction. Thus, we suggest that at least the synthesis of high-molecular-weight mRNA-like RNA starts already at the first cleavage and the rate per nucleus is probably constant during cleavage. This rate may increase lo- to loo-fold at the time of the midblastula transition. Interesting in this connection is a report by Gurdon and Woodland (1969). These authors noticed by sucrose density gradient analysis the slight RNA labeling in early cleavage embryos injected with 3H-labeled nucleosides. However, these authors could not detect any

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