Synthesis and turnover of polysomal mRNAs in sea urchin embryos

The synthesis and turnover kinetics of polysomal mRNA have been measured in sea urchin embryos. Polysomes were isolated from stages ranging between mesenchyme blastula and late gastrula Strongylocentrotus purpuratus embryos which had been exposed to exogenous ³H-guanosine. The amount of radioactivity incorporated into messenger and ribosomal RNAs was determined separately as a function of time, and the precursor pool specific activity was measured in the same embryos. Synthesis and decay rate constants were extracted from the data by a leastsquares procedure. Per embryo, the rate of mRNA synthesis was calculated to be about 0.13 pg min^?1, while the rate of rRNA synthesis is about 0.022 pg min^?1. The newly synthesized mRNA turns over with a half-time of 5.7 hr. The data support only a single decay rate for the mRNA, but small fractions of mRNA decaying at different rates cannot be excluded. Previous studies have shown that a minor fraction of the mRNA includes the least abundant, most highly diverse set of messages (“complex class” mRNAs). To determine whether mRNAs of the complex class are synthesized and degraded at similar rates, labeled mRNA was measured in hybrids formed in mRNA excess reactions with single copy DNA. These experiments showed that complex class mRNAs represent an approximately proportional amount of the new mRNA synthesis, and turn over at the same average rate as does the bulk of the mRNA. Most of the mRNAs in the embryo polysomes are newly synthesized, rather than maternal. This statement refers both to complex class mRNAs and to prevalent mRNAs. Considering the sequence homology between embryo and oocyte mRNAs shown earlier, these results indicate that many of the same structural genes active during oogenesis are being transcribed in embryos at these stages.     

Chromatin proteins of sea urchin embryos: Dual origin from an oogenetic reservoir and new synthesis

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.     

Nuclear histones of unfertilized sea urchin eggs

Histones have been isolated from the nuclei of unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The electrophoresis of these histones exhibits a pattern different from that of the sperm or embryo of the same species.     

Species-specific dissociation into single cells of live sea urchin embryos by fab against membrane components of Paracentrotus lividus and Arbacia lixula

The species and stage specificities of membrane components active in promoting reaggregation of cells dissociated from embryos of the two Mediterranean sea urchin species Paracentrotus lividus and Arbacia lixula have been examined. Membrane proteins extracted with butanol either from purified membranes or from dissociated cells without significant reduction of viability promoted reaggregation of both the homologous and heterologous species. Extracts from plutei and blastulae were equally effective in promoting reaggregation of blastula cells. By contrast, Fab's prepared from IgG raised against these extracts or purified membranes are strictly species specific because they prevent reaggregation of cells and actively dissociate live embryos of only the homologous species. No corresponding stage specificity of the Fab was observed: Fab against extracts from blastula embryos also caused dissociation of plutei. Antigenic analysis of the extracts by the Ouchterlony test revealed the presence of components specific for each species as well as others common to both.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein

Indirect immunofluorescence staining of cleaving sea urchin eggs with an antiserum against a tryptic fragment of dynein 1 (fragment 1A) from sea urchin sperm flagella suggested the presence of dynein in the cortex as well as in the mitotic apparatus. In the present study, we found that the Mg²⁺-ATPase activity of the isolated cortices from sea urchin eggs, which exhibited similar characteristics to those of flagellar dynein, was inhibited by 60–80% with the anti-fragment 1A serum. Faintly stained bands corresponding to the A-band (dynein 1) and the B-band of the sperm flagella was detected on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the isolated cortices. Furthermore, the SDS-gel electrophoresis revealed the presence of a polypeptide band corresponding to dynein 1 in the antigen-antibody complex precipitated from the KCl-extract of the cortices with the antiserum.     

Heterogeneous nuclear RNA (HnRNA) size in developing frog embryos.

Almost one-fifth of cells disaggregated from 16-cell mouse embryos neither divide nor die. On the basis of a number of criteria, it is concluded that these cells differentiate to trophoblast. Thus, neither cell-cell interaction nor cell division is required for execution of the programme for trophoblast differentiation from the 16-cell stage.     

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.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).     

Timing, localization, and control of wheat germ agglutinin synthesis in developing wheat embryos

The lectin, wheat germ agglutinin (WGA), is synthesized de novo by developing wheat (Triticum aestivum, L.) embryos but is not synthesized or localized in developing endosperm as shown by radioimmunoassay. Young embryos removed from the grain and cultured on a defined medium germinate precociously and concomitantly cease WGA synthesis. In vitro precocious germination of young embryos is reversibly inhibited by low levels (1–100 μM) of the plant growth substance abscisic acid (ABA). Embryos inhibited from germinating by this growth regulator not only continue synthesizing WGA, but do so at an accelerated rate when compared with embryos left associated with the grain.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Similarity of proteins synthesized by isolated blastomeres of early sea urchin embryos

The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [³⁵S]methionine until the next cleavage. Nearly all the radioactive protein was solubilized and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1000 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.