Mouse kidney nonpolysomal messenger ribonucleic acid: metabolism, coding function, and translational activity

To elucidate the distribution and function of mRNA in mouse kidney cytoplasm, we compared mRNA isolated from polysomal (greater than 80S) and native postpolysomal (20--80S) ribonucleoproteins with respect to synthesis and lifetime, sequence content, and translational activity. The 20--25% of cytoplasmic mRNA recovered from postpolysomal ribonucleoprotein is similar to polysomal mRNA in size (20--22S), in apparent half-life (11--13 h), in major products of cell-free translation, and in nucleotide complexity (approximately 4 x 10(7) nucleotides). The labeling kinetics of polysomal and postpolysomal mRNA suggest these mRNA populations are in equilibrium. [3H]cDNAs transcribed from polysomal and from postpolysomal poly(A)-containing mRNAs react with template mRNA and with the heterologous mRNA at the same rate (Cot1/2 approximately 6.3 mol.s/L) and to the same extent (95%). Therefore, these mRNAs are equally diverse and homologous and occur at similar relative frequencies. Postpolysomal mRNA directs cell-free protein synthesis at only approximately 30% of the rate of polysomal mRNA and to only 30% of the extent of mRNA from polysomes. Postpolysomal mRNA is approximately 3-fold less sensitive than polysomal mRNA to inhibition of translation by m7GMP, suggesting postpolysomal mRNA contains a greater fraction of molecules deficient in 5'-terminal caps. Postpolysomal mRNA may derive from renal mRNAs that initiate translation inefficiently and thus accumulate as postpolysomal ribonucleoproteins.     

Electron microscope visualization of giant polysomes in sea urchin embryos

Chromatin spreading techniques have been applied to the electron microscopic visualization of polysomes in sea urchin (Strongylocentrotus purpuratus) eggs and embryos. Polysomes of giant size are commonly found after the 8-cell stage. The largest seen, from an early gastrula, was 13.6 m in length, carried 277 ribosomes, with a message calculated to contain 6.49×10⁴ nucleotides and potentially to encoded 2.38×10⁶ daltons of peptide. Polysomes are rare and very large ones absent from lysates of unfertilized eggs. Giant polysomes appear in 4- to 8-cell stages and are common in 16-cell stages and thereafter. They are of two forms: a compact form with no spacing between ribosomes characteristic of stages through early mesenchyme blastulae, and an extended form found only after late mesenchyme blastulae. Both have potential for massive informational content. Some of each type have ribosome-free tails at one end, as long as 733 Å in the compact forms, and 7,890 Å in the extended ones. Occasionally they have a single array of fibrous material increasing from one end of a polysome to the other, interpreted to be nascent peptide chains. Polysomes are not found after brief, mild exposure of lysates to RNase A, or from embryos treated with puromycin. Very large polysomes are present in lysates of blastulae exposed since fertilization to actinomycin D, cycloheximide, or cordycepin. They appear in parthenogenetically activated or fertilized enucleate merogones, but are absent from unactivated merogones, demonstrating that egg masked messages can generate them. A potential embryological significance of giant, potentially polycistronic polysomes is suggested.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Nucleic acid and histone synthesis by ethanol-treated cleavage-arrested sea urchin embryos

It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanol-treated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20-30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.