On the size relationship between nuclear and cytoplasmic RNA in sea urchin embryos

The approximate sizes of heterogeneous nuclear (HnRNA) and cytoplasmic RNA of sea urchin embryos were determined by DMSO density gradient centrifugation and acrylamide-formamide gel electrophoresis. The data suggest that the sizes of these molecules are smaller than those estimated under nondenaturing conditions. The size of most of the nuclear RNA ranges from 0.5 to 3 × 10⁶ daltons, while that of the cytoplasmic RNA ranges from 0.1 to 2 × 10⁶ daltons. Both nuclear and cytoplasmic RNA of sea urchin embryos may have a minor fraction (5–10%) of very large species with molecular weights up to 4 to 5 × 10⁶ daltons.The idea that the size of HnRNA may be larger in organisms higher on the evolutionary scale is discussed.     

Oligomeric sequences in the cytoplasmic RNA of sea urchin embryos

Oligomeric stretches of adenylate and uridylate and polymeric segments of adenylate have been shown to exist in sea urchin embryo hnRNA. It is demonstrated here that at least some oligo(U)-enriched sequences are conserved in sea urchin cytoplasmic RNA, whereas apparently few, if any, oligo(A) sequences are so conserved.     

Molecular classes of heterogeneous nuclear RNA in sea urchin embryos.

Nucleotide sequences within a phage genome can be detected in individual phage plaques by in situ hybridization with complementary RNA sequences. Results with phage A and a derivative having 10% of its DNA deleted indicate that sequences 500 to 1000 base-pairs long should be detectable with confidence.     

A cytoplasmic dynein heavy chain in sea urchin embryos.

By making the hypothesis that the pattern of conserved sequence residues in the vicinity of the hydrolytic ATP-binding site of dynein would resemble that in myosins from a broad variety of sources, we designed degenerate oligonucleotide primers capable of amplifying this region of multiple dynein isoforms from sea urchin embryo poly(A)+ RNA. Quantification of the expression of two of these dynein isoforms has shown that the level of mRNA encoding for the beta-heavy chain, like that of tubulin, increases 2-3-fold after deciliation of the embryos, whereas the expression of the second dynein isoform, like that of actin, is essentially unaffected. This second isoform is believed to be the cytoplasmic dynein of sea urchin embryos.     

Small molecular weight RNA components in sea urchin embryos

Polyacrylamide gel electrophoresis of RNA from Paracentrotus lividus embryos has shown this material to contain five RNA components of small molecular weight. The components are synthesized early in sea urchin development, simultaneously with tRNA and heterodisperse RNA. After the blastula stage, when synthesis of ribosomal RNA is activated, the labeling incorporated into small molecular weight RNA components constitutes a relatively decreasing proportion of the total labeling in RNA. When labeling is performed prior to the blastula stage, three of the small molecular weight RNA components are labeled to a similar or greater extent than “5” S RNA and the 26-ass RNA. The gel electrophoretic mobilities of the small molecular weight RNA components have been compared with those in Ehrlich ascites cells and found to be different.     

Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos.

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.     

Synthesis of RNA in isolated nuclei of sea urchin embryos

Summary It is demonstrated that isolated nuclei of sea urchin embryos are able to incorporate radioactive nucleotides into RNA.Some properties of the incorporation system are described.     

Rates of RNA chain growth in developing sea urchin embryos

The average time necessary to add a nucleotide onto growing RNA chains (step time) has been determined for several developmental stages of Strongylocentrotus purpuratus embryos. One procedure involved quantitation of isolated nucleosides derived from the 3′ end of newly synthesized chains plus total rates of nucleotide incorporation. The former values provide the number of growing chains so that rates of incorproation per RNA chain may be calculated. A second procedure involves determinations of the times required for RNA molecules of given size classes to become fully labeled. These times may be equated to the synthetic times for the particular size class [Bremer, H., and Yuan, D. (1968). RNA chain growth-rate in Escherichia coli. J. Mol. Biol.38, 163–180]. The step times for blastula and pluteus embryos is 6–9 nucleotides/sec at 15°C, which would result in an average synthetic time for heterogeneous nuclear RNA (HnRNA) molecules of about 2 × 10⁶ daltons of 12–18 min. The half-life of HnRNA in this species of sea urchins is 15–20 min, implying a transient existence for large HnRNA molecules.