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.     

Carbonic anhydrase activity in developing sea urchin embryos

A 13-fold increase in carbonic anhydrase specific activity was found during the first 24 h in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Carbonic anhydrase activity was sensitive to inhibition by 10⁻⁴ M acetazolamide. Roles for carbonic anhydrase activity in intracellular pH regulation and spicule formation are discussed.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

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.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

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.     

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.     

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.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

Regulation of synthesis of the larval serum proteins of Drosophila melanogaster

We have determined the relative amounts of subunits of larval serum proteins (LSPs) 1 and 2 during larval development in Drosophila melanogaster. These results indicate that synthesis of polypeptide subunits of LSP-1 and LSP-2 is coordinate: the proteins are first detected at the same time; they accumulate in a coordinate fashion; their RNAs are first detected at the same time; the RNAs also accumulate in similar relative amounts. Analyses of fat body polypeptides and fat body RNA indicate that synthesis of LSP-1 declines at a time when there are still substantial quantities of LSP-1 RNA in the cytoplasm. Cessation of LSP-1 subunit synthesis occurs before cessation of LSP-2 synthesis, indicating that at late times the genes (or mRNAs) for these two proteins are subject to different "switch-off" controls.