Y. Misumi

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.

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

SummaryThe mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)+ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)+ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)+ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.

RNA metabolism in primary cultures of Xenopus laevis kidney cells: III. Processing of rRNA precursor in growing and resting cells

Abstract The rate of processing of ribosomal RNA (rRNA) precursor was measured in primary cultures of Xenopus laevis kidney cells in growing and resting states. The kinetic studies on labeling of the precursor RNA and 18 and 28S rRNA in cumulative and pulse-chase experiments revealed that the precursor accumulated and reached a steady state much more rapidly and 18 and 28S rRNA started to appear more rapidly in actively growing cells. Furthermore, the halflives of the precursor RNA were estimated to be 10 and 30 min for growing and resting cells, respectively. These results clearly support the view that not only the transcription and breakdown which have already been shown to be important steps for regulation, but the processing of the precursor RNA molecule would also be controlled upon alteration of the cellular growth rate.

Does the rate of ribosomal RNA synthesis vary depending on the number of nucleoli in a nucleus

Abstract Cultured kidney cells of Xenopus laevis were pulse-labeled with [ 3 H]uridine for 10, 20 and 30 min during their logarithmic growth phase and then processed for autoradiography. The labeled cells were assigned into two categories, one- and two-nucleolated cells, and the rate of ribosomal RNA (rRNA) synthesis was measured by counting the number of grains in nucleoli. The results obtained revealed that a two-nucleolated cell incorporated significantly much more radioactivity into its nucleoli than did a one-nucleolated partner for all the periods examined. Cells of these different nucleolar types, however, contained essentially the same amount of rDNA (DNA complementary to rRNA) as estimated by in situ hybridization with [ 125 I]rRNA. Although it remains to be proved that the observed increase in incorporation represents the increased rate of rRNA synthesis in two-nucleolated cells, the present findings seem to be very interesting, since they might indicate that the activity of rRNA genes is in some way regulated or affected by their spatial relationship in a cell nucleus.

INITIATION OF RIBOSOMAL RNA SYNTHESIS AND CELL DIVISION IN XENOPUS LAEVIS EMBRYOS

Isolated cells from animal or vegetal pole regions of Xenopus blastulae were cultured, and the timings of rRNA synthesis and cell division were determined. rRNA synthesis was measured by the incorporation of (3H)methionine into rRNA, and cell division was studied by the decrease in cell size and rRNA content. Nucleoli in these cells were also examined. It was found that these animal and vegetal cells continue to divide under the present conditions in the same temporal pattern as that of intraembryonic cells, and that rRNA synthesis begins soon after the cells have undergone the division which probably corresponds to the 15th division following fertilization. At this time, the rRNA content and concentration of the animal and vegetal cells are significantly different. These results seem to support the view that cell division is involved in some way in the mechanism determining the timing of rRNA synthesis in early embryonic development.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos: VI. Occurrence in mature oocytes and unfertilized eggs

Abstract Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis . It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [ 3 H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.