Koichiro Shiokawa

Pattern of RNA synthesis in isolated cells of Xenopus laevis embryos

Abstract It has been shown that in isolated cells obtained from Xenopus laevis embryos, uridine- 3 H, thymidine- 3 H, and 14 C-labeled protein hydrolyzate are incorporated very rapidly into acid-insoluble materials. The extent of incorporation is strikingly greater than that into intact embryos. The uridine- 3 H incorporation is sensitive to actinomycin D at a concentration as low as 0.5 μg/ml. These isolated cells derived from blastulae have been shown to synthesize soluble and ribosomal RNA in an orderly succession, as do intact embryos. The synthesis of soluble RNA is followed by that of ribosomal RNA, the latter being initiated only after incubation for a certain period. During this period, the intact embryos reach the gastrula stage and begin ribosomal RNA synthesis. The synthesis of ribosomal RNA in the isolated cells is then activated very rapidly. It has been suggested on the basis of the results obtained that the isolated cells can be used for biochemical investigations.

A study on the steady-state population of poly(A)+RNA during early development of Xenopus laevis☆

The steady-state population of poly(A)-containing RNA [poly(A)+RNA] was studied from oogenesis to organogenesis of Xenopus laevis, utilizing [3H]poly(U) hybridization combined with sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. Throughout the developmental stages examined, almost all poly(A) sequences detected were not free but were associated with heterogeneous RNA which had a modal sedimentation constant of 18 S or less. During oocyte maturation and ovulation, the poly(A) content decreased from 1.8 ng per oocyte to 1.0 ng per egg. After fertilization, it increased again to 1.7 ng per embryo at the blastula stage, decreased slightly during gastrulation and then finally increased continuously to 6 ng at the heartbeat stage. Changes in the content and number of poly(A)+RNA molecules roughly paralleled the poly(A) content. Poly(A)+RNA contained two distinct size classes of poly(A) in oocytes (mean length; 60 and 20 nucleotides long) but three in eggs and embryos (100, 80, and 20 nucleotides). The proportion of the RNA which contained poly(A)100 became larger after the blastula stage. Cell fractionation revealed that poly(A)100+RNA was localized in both the nucleus and cytoplasm, poly(A)80+RNA mainly in the nucleus, and poly(A)20+RNA exclusively in the subribosomal fraction of embryonic cells. Labeling experiments showed that poly(A) synthesis occurring in the cleavage cells was not affected at all by the inhibition of RNA transcription, while it was abolished greatly in the postblastula cells. The newly synthesized poly(A) sequences were found to be associated with preexisting and newly synthesized heterogeneous high molecular weight RNA in the cleavage and postblastula cells, respectively. Our present findings, taking the published data into account, can be taken as indication of (1) possible deadenylation of a population of maternal poly(A)+RNA during oocyte maturation, (2) polyadenylation of the same or a different RNA population after fertilization, (3) possible destruction of maternal RNA during gastrulation, and (4) predominancy of newly synthesized RNA after the neurula stage.

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

It has been proposed and is now widely accepted that in Xenopus laevis 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 Xenopus embryos. In the present experiments, we cultured fertilized eggs in 80 mM phosphate buffer and loosened the adhesion between blastomeres, so that [3H]uridine could be incorporated into blastomeres from the surrounding medium. By this method and also by microinjection of [3H]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 alpha-amanitin. From these results we conclude that mRNA-like RNA and low-molecular-weight RNA start to be synthesized during the cleavage stage.

Demonstration of rRNA Synthesis in Pre‐Gastrular Embryos of Xenopus Laevis

Isolated cells from Xenopus laevis embryos at various developmental stages were incubated with (methyl‐3H) methionine to label newly synthesized RNAs. Methylation of RNAs was studied by analyzing nuclease‐digests of high‐molecular‐weight RNAs by DEAE‐Sephadex column chromatography. Labeled rRNA, mRNA and 4s RNA were distinguished by their characteristic patterns of 2′‐0‐methylation and base‐methylation. It was concluded that rRNA synthesis starts during the mid‐ to late‐blastula stage. This is about 4 hr, or at least 3 cell cycles earlier than the initiation of gastrulation, which was previously considered to be the stage when rRNA synthesis begins.

Non‐Coordinated Synthesis of RNA's in Pre‐Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre‐ and post‐gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 104 molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis

Abstract Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos

A fraction containing acid-soluble materials (PCA-extract) from Xenopus early blastulae preferentially inhibits the incorporation of [3H]uridine into 18S and 28S rRNA in Xenopus neurula cells. Pulse- laneling experiments revealed that whereas tubercidin, a known inhibitor of rRNA processing, produced a marked accumulation of the label in 40S pre-rRNA, Xenopus inhibitor suppressed labeling of the pre-rRNA. When tubercidin was added to cells whose activity for rRNA synthesis had been lowered by the Xenopus inhibitor, there was still an accumulation of label in the pre-rRNA. These results indicate that Xenopus inhibitor suppresses the synthesis of rRNA at transcription rather than at processing.

Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis

We analyzed the accumulation of newly-synthesized heterogeneous mRNA-like RNA, 4 S RNA, 5 S RNA, snRNAs and rRNA before and after the midblastula transition (MBT) in Xenopus laevis embryogenesis. Based on the kinetics of the labeling, we concluded that the pattern of RNA synthesis in Xenopus embryogenesis changes following at least three characteristically different phases. The first phase is the pre-MBT stage, which is characterized by the synthesis of heterogeneous mRNA-like RNA, accompanied by the synthesis of small amounts of 4 S RNA, 5 S RNA and snRNAs. The second phase is the MBT stage which is characterized by a large activation (about 50-fold increase on a per cell basis) of 4 S RNA synthesis. The third phase is the post-MBT stage which is characterized by the commencement and increase in rRNA synthesis. We assume that RNA polymerases II, III and I are activated in this order in early Xenopus embryogenesis.

Expression of exogenously introduced bacterial chloramphenicol acetyltransferase genes in Xenopus laevis embryos before the midblastula transition

SummaryPrevious papers have reported that DNAs exogenously injected into Xenopus laevis fertilized eggs are expressed only at and after the midblastula transition (MBT). We have injected fertilized eggs of Xenopus laevis with circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes connected to the promoter of viral genes (pSV2CAT and pAd12.E1aCAT) or the Xenopus cardiac actin gene (actin-CAT fusion gene), and examined whether these DNAs are expressed during the stage before the MBT. We found that expression of CAT enzyme can be detected before the MBT when CAT genes connected to viral promoters were injected. The activity was low during the cleavage stage on a per-embryo basis; however, the time course of the accumulation of the CAT enzyme activity roughly paralleled the increase in cell number. Therefore, CAT enzyme activity per cell was constant during cleavage and did not change dramatically before and after the MBT stage. CAT mRNA level, detected by CAT antisense RNA, was roughly proportional to the levels of CAT enzyme. Therefore, we assume that the observed enzyme activity reflects the transcriptional activity of injected CAT genes before and after the MBT. When the actin-CAT fusion gene was injected, however, no enzyme activity was detected until embryos had reached the neurula stage, a stage when endogenous actin genes are first activated. On the basis of these results, we conclude that the concept of an initial transcriptional activation of exogenous genes at amphibian MBT has to be changed. We propose that the expression of polymerase-II-transcribed genes is regulated by the nature of the promoters connected to the genes rather than by changes associated with MBT.

DIFFERENTIAL INITIATION OF rRNA GENE ACTIVITY IN PROGENIES OF DIFFERENT BLASTOMERES OF EARLY XENOPUS EMBRYOS: EVIDENCE FOR REGULATED SYNTHESIS OF rRNA

The dorsal and ventral blastomeres of 4‐celled embryos of Xenopus luevis were separated. During the next 14 hr in cultnre, the cell numbers of the progeny cell aggregates of the dorsal and ventral blastomeres respectively, were the same. Synthesis of 4 S RNA started after about 4 hr of culture in both kinds of progeny cell aggregates. However, a clear‐cut difference was found in the time when rRNA synthesis started in these aggregates: it began after 10 hr in cell aggregates derived from the dorsal blastomeres, and after about 14 hr in those derived from the ventral blastomeres. After this, rRNA synthesis became more and more active in both types of aggregates. This is the first demonstration of differential initiation of rRNA synthesis in aggregates derived from different blastomeres but containing the same number of cells. The results provide unequivocal evidence for developmental regulation of rRNA synthesis.