The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Simultaneous synthesis,translation, and storage of mRNA including histone mRNA in sea urchin eggs

The kinetics of accumulation of RNA labeled with uridine and the time course of change in the specific activity of the UTP pool were used to estimate the rate constants for synthesis and decay of RNA synthesized in unfertilized eggs of the sea urchin Lytechinus pictus. The rate of synthesis per haploid genome is similar to that in embryos. Most of the RNA is turning over with a half-life of about 5 hr, and an average of 11 pg of newly synthesized RNA accumulates at steady state. About 3.7% of the RNA in the polysomes of the egg is newly synthesized and this RNA has the heterogeneous size distribution expected for mRNA. Thus most, probably all, of the mRNA translated in the egg is also synthesized in the egg. Little, if any, of the RNA synthesized in the egg enters polysomes following fertilization. Thus the egg synthesizes a population of mRNA which is unstable and translated, but it also contains a more stable, untranslated population of previously synthesized, stored mRNA, which is translated only after fertilization. Since the two populations of mRNA code for the same abundant proteins (Brandhorst, B. P. (1976). Develop. Biol., 52, 310–317), there is a temporal separation in the metabolism and function of coexisting mRNA molecules of identical coding sequence. Among the mRNAs synthesized and translated in the egg are histone mRNAs having the same electrophoretic mobilities and rates of synthesis per genome as those synthesized in rapidly cleaving embryos. Thus the synthesis, entry into the cytoplasm, and translation of histone mRNA are not restricted to the S phase of the cell cycle or the period of cell division.     

Re-examination of histone changes during development of newt embryos

Embryos of Triturus pyrrhogaster (BOIE) were labeled with Na₂¹⁴CO₃ and the incorporation of radioactivity into histone fractions was determined by the electrophoresis of the acid-soluble protein from isolated nuclei on a polyacrylamide gel with or without Triton X-100. The results supported the previous observation that the content of H1 histone might be low in blastulas and increased during development but they did not confirm the displacement of blastula H1 by other H1 molecular species in later embryos. The rate of H2b or H2a histone synthesis did not change much during development which contrasted sharply with the case of histone synthesis in sea urchin embryos. By changing the label duration or by culturing various durations after the label it was suggested that the histone fractions were synthesized or degraded as a set and any particular fraction that had a markedly long or short life could not be detected. The results were discussed in relation to the possible functions of H1 histone and to the histone synthesis in sea urchin embryos.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

DNA sequences flanking the starts of the hsp 70 and alpha beta heat shock genes are homologous

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26°C. The extent of this delay varied with incubation pH, but not with concentrations of K⁺, Mg²⁺, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.     

Effects of cytochalasin B on sperm-egg interactions

The effects of cytochalasin B (CB) on the interaction of sea urchin (Arbacia), molluscan (Spisula), and mammalian (Mus) gametes have been examined. Despite the absence of sperm incorporation and gamete membrane fusion, CB-treated Arbacia and Spisula eggs (1–10 μg/ml for 1–10 min) mixed with sperm activated. Unlike the situation observed in Arbacia and Spisula, mouse eggs treated with CB (1–50 μg/ml for 1 hr) are capable of sperm incorporation. These data are discussed with reference to possible mechanisms by which sperm may induce eggs to activate.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

The Relative Contributions of Newly Synthesized and Stored Messages to Hl Histone Synthesis in Interspecies Hybrid Echinoid Embryos

We have measured the ratio of incoropation of ³H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histone mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA respresents 80% of total Hl histone synthesis at the 16–32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation.
These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

Synthesis of rabbit globin in a cell-free protein synthesis system utilizing sea urchin egg and zygote ribosomes

Reports of the reduced ability of sea urchin egg ribosomes to participate in synthetic mRNA-directed protein synthesis have fostered the suggestion that the low protein synthesis rate of eggs is due to ribosome-associated inhibitors. To test this hypothesis with a natural message, we have isolated 80S ribosomes and microsomal ribosomes of sea urchin eggs and zygotes and compared their activity at synthesizing protein from rabbit α and β globin mRNA in a Krebs II ascites tumor cell-free system. Both egg and zygote 80S ribosomes responded to added mRNA and were shown to synthesize complete α and β globin chains by CM-cellulose chromatography. In most cases, the activity of the egg ribosomes was in comparable instances higher than the zygote ribosomes. Attempts to determine the cause of this difference indicated that it was not a function of K⁺ or Mg²⁺ concentration, type of tRNA used, or ribosomal wash proteins. From these studies it is apparent that sea urchin egg ribosomes are functional at a level equivalent to or better than zygote ribosomes, and it appears that the lack of protein synthetic activity in unfertilized eggs is not due to the presence of a population of inhibited ribosomes.     

The relative contributions of newly synthesized and stored messages to Hl histone synthesis in interspecies hybrid echinoid embryos.

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

Acid release following activation of surf clam (Spisula solidissima) eggs.

Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.     

A test for masked message: the template activity of messenger ribonucleoprotein particles isolated from sea urchine eggs

The hypothesis that the “masked message” of unfertilized eggs consists of nontranslatable mRNP particles was directly tested by in vitro translation of mRNPs in a system derived from wheat germ. Three classes of mRNPs were tested: particles prepared from sea urchin eggs in buffers containing 0.35 M K⁺, particles prepared from sea urchin eggs in 0.35 M Na⁺, and particles released with EDTA in 0.35 M K⁺ from polysomes of sea urchin embryos cultured in the presence of actinomycin D. The mRNA content of particles was monitored by determination of poly(A) content. The wheat germ system used is quantitatively stimulated by addition of mRNA derived from eggs or from any of the classes of mRNPs used. Particles prepared from eggs with Na⁺ or released from polysomes contain less protein than particles isolated from eggs in K⁺, and as expected these particles are fully translatable in vitro. Particles prepared from eggs in buffers containing 0.35 M K⁺ produce little or no stimulation in the in vitro system. That this lack of translation represents in vivo masking is indicated by several considerations: (1) The nontranslatable particles were prepared in 0.35 M K⁺ and 5 mM Mg²⁺, ion concentrations similar to those found in echinoderm eggs; (2) density and sedimentation rate characteristics of the particles are little changed by isolation; (3) RNA extracted from isolated particles is fully translatable; and (4) particles prepared from polysomes or under conditions which destabilize RNPs are translatable. These data support the masking hypothesis for the protein synthesis repression system of eggs.     

Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus

The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage (CS) variants. However, after fertilization, both CS and alpha messages are translated. Since alpha histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of alpha histone subtypes appears to be under translational control. Although the synthesis of alpha subtypes is shown here to occur before the second S phase after fertilization, little or no alpha histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.