Characterization of pulse-labeled nuclear RNA in sea urchin embryos

doi:10.1016/0014-4827(72)90592-7

Experimental Cell Research

Volume 72, Issue 1, May 1972, Pages 309-324

https://doi.org/10.1016/0014-4827(72)90592-7 Get rights and content

Abstract

Pulse-labeled RNA in sea urchin embryos is largely confined to the nucleus even after removal of membranes with detergents. Various reconstruction controls eliminated extensive cytoplasmic contamination of the nuclear preparations although a small amount of contamination by ribosomes or polyribosomes could not be excluded. The RNA was present in very large aggregates following solubilization by treatment of nuclei with detergents plus sonication. These aggregates were degraded by treatment with Pronase or ribonuclease but not by deoxyribonuclease. Sensitivity to EDTA was only partial and dependent on the detergent used for solubilization. Synthesis and transport to the cytoplasm of pulse-labeled RNA was not inhibited by puromycin. After puromycin treatment the nuclear RNA was still present in aggregates and the cytoplasmic RNA was distributed in polyribosomes, just as in the control.

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Changes in the synthesis and intracellular localization of nuclear proteins during embryogenesis in the sea urchin Strongylocentrotus purpuratus
1987, Developmental Biology
A rapid, gentle technique is described for the isolation of nuclei from sea urchin embryos. Using this technique, we have analyzed the synthesis and accumulation of nonhistone nuclear proteins during sea urchin development by two-dimensional gel electrophoresis. Most nuclear proteins fall into one of three patterns of synthesis, which are distinguished by maximal rates of accumulation at early (prior to hatching blastula), middle (hatching blastula/gastrula), or late (prism/pluteus) stages of development. Over 60% of observed nuclear proteins undergo apparent qualitative changes in synthesis and accumulation between the 64-cell and pluteus stages. Most of these changes represent appearances of new proteins. A large number of qualitative changes occur very early in development; the period of greatest change is between the 64-cell and 200-cell stages. Over half of the proteins which first appear in the nucleus subsequent to the 64-cell stage are synthesized at stages prior to the time of their initial appearance in nuclei, but are excluded from nuclei for some time.
Chromatin proteins of sea urchin embryos: Dual origin from an oogenetic reservoir and new synthesis
1981, Developmental Biology
The chromatin proteins of different embryonic stages, ranging from 16 cell to gastrula, of the sea urchin Strongylocentrotus purpuratus were labeled, in vivo, with ¹⁴C and were labeled, in vitro, with ³H. The proteins thus labeled were separated by high resolution two-dimensional electrophoresis. The extent of possible cytoplasmic contamination has been examined with reconstruction experiments. Gastrula chromatin contains over 200 separable nonhistone proteins, and about 90% of them are also detected at the 60-cell stage; cleavage stages have over all protein gel patterns displaying numerous differences with the pattern shown by chromatin from later stages. Differences in the proportion of histone to nonhistone proteins that are synthesized are observable at the different embryonic stages, with histones predominating in midcleavage. About half of the nonhistone proteins of the developing embryo that can be labeled with ³H, in vitro, are not labeled with ¹⁴C, in vivo, and hence, must originate from a reservoir of nonhistone proteins assembled during oogenesis.
Change in transcription patterns during early development of the sea urchin
1978, Developmental Biology
Complementary DNA (cDNA) probes complementary to polyadenylated mRNA and HnRNA from sea urchin blastulas were synthesized by reverse transcriptase to analyze changes in prevalent class RNA during early development. About 17% of A⁺HnRNA and 62% of A⁺mRNA is transcribed into the cDNA probes. As measured by RNA-excess hybridization, the complexity of cDNA transcribed from HnRNA was 4.1 ± 1 × 10⁷ nucleotides, and the complexity of cDNA from mRNA was 6.0 ± 1.2 × 10⁵ nucleotides. These values correspond to 12 to 20% and 2 to 3% of the total blastula HnRNA and mRNA complexity, respectively. The cDNA thus represents a limited subset of the total mRNA sequences but a substantial fraction of the HnRNA sequences. The cDNA probes were hybridized with a large excess of the corresponding polyadenylated RNA populations from gastrula and pluteus embryos. Plateau values appeared to be substantially lower for the heterologous reactions. The kinetics of the reactions indicate a two- to threefold reduction in abundance of HnRNA species that persist to gastrula and a further small reduction at pluteus. The abundance of the persistent mRNA transcripts decreases 20-fold at gastrula but remains constant thereafter.
Alterations of RNA metabolism in sea urchin embryos by an inhibitor of protein synthesis initiation
1977, BBA Section Nucleic Acids And Protein Synthesis
An inhibitor of the initiation of protein synthesis, $2-(4 methyl -2,6 dinitroanilino)-N- methyl$ propionamide, (MDMP), inhibits incorporation of amino acids by 85–95% without altering the rate of incorporation of uridine into sea-urchin-hatched blastula embryos. Since the inhibition is readily reversible up to at least 90 min after addition, extensive secondary effects are unlikely. Newly synthesized RNA accumulates in polyribosome-like structures in amount and with a size distribution very similar to the control. The polyadenylation of total, cytoplasmic and polyribosomal RNA are only slightly different from the controls. While the size distribution of 9-S to approx. 30-S RNA is about the same, there is relatively less low molecular weight RNA (approx. 4-S). There is also a greater accumulation of $>30- S$ RNA in the cytoplasm of treated embryos due either to leakage and/or improper processing of nuclear RNA. The latter possibility is consistent with the decrease in 4-S RNA which is a presumed degradation product of nuclear HnRNA. While the initial rate of RNA synthesis and much of the accumulation of RNA in polyribosomes are not affected, the inhibition of protein synthesis per se or a secondary effect of MDMP results in altered patterns of RNA transport and processing.
Rates of RNA chain growth in developing sea urchin embryos
1977, Developmental Biology
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.
Sequence complexity of the RNA accumulated in oocytes of Arbacia punctulata
1976, Developmental Biology
The sequence complexity of the RNA present in mature oocytes of the sea urchin Arbacia punctulata was measured by DNA-RNA hybridization. Small quantities of single copy [³H]DNA were hybridized with large excesses of RNA and the amount of [³H]DNA-RNA hybrids formed was assayed by hydroxyapatite. At termination of the reaction, which follows pseudo-first-order kinetics, 2.6% of the reactable single copy [³H]DNA mass is present in RNA-DNA hybrids. This DNA was isolated, shown to be rehybridizable to RNA and, within the range of detectability, to consist solely of nonrepetitive sequences. Assuming asymmetric transcription, 5.2% of the genomic single copy sequence, or 3.0 × 10⁷ nucleotides, is represented in oocyte RNA. The kinetics of hybridization indicate that these sequences are included in approximately 1–2% of the oocyte RNA.

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Characterization of pulse-labeled nuclear RNA in sea urchin embryos

Abstract

J mol biol

J biol chem

J mol biol

Exptl cell res

Virology

Dev biol

Biochem biophys res commun

J biol chem

J mol biol

J mol biol

J mol biol

J biol chem

Anal biochem

J mol biol

J mol biol

Dev biol