Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): mapping of homologies in coding and spacer DNA

The linear arrangement and lengths of the spacers and coding regions in the two nonallelic histone gene variant clusters of L. pictus are remarkably homologous by R loop analysis and are similar in general topography to the histone gene repeat units of other sea urchins examined to date. No interventing sequences were detected. The coding regions of these two histone gene variants share considerable sequence homology; however, there are areas of nonhomology in every spacer region and the lengths of the nonhomologous spacers between the H2A and H1 genes are not the same for the two repeat unit classes (inter-gene heterogeneity). Combining length measurements obtained with both R loops and heteroduplexes suggests that the DNA sequences of the analogous leader regions for the two H1 mRNAs are nonhomologous. Similar observations were made for the H4 leader sequences, as well as the trailer region on H2B. S. purpuratus spacer DNA segments share little sequence homology with L. pictus; however, the analgous coding (and possibly flanking) regions have conserved their sequences. The various coding and spacer regions within a repeat unit do not share DNA sequences. Thus certain areas in the sea urchin histone gene repeat units have been highly conserved during evolution, while other areas have been allowed to undergo considerable sequence change not only between species but within a species.     

Evidences of two different sets of histone genes active during embryogenesis of the sea urchin Paracentrotus lividus.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

The early and late sea urchin histone H4 mRNAs respond differently to inhibitors of DNA synthesis

The effect of inhibiting DNA synthesis on the concentration of the alpha-histone mRNA and the late histone mRNAs in sea urchin embryos was measured. The alpha-histone mRNA concentrations did not change, while the late histone mRNA concentrations were rapidly reduced at the three developmental stages (morula, blastula, and mesenchyme blastula) tested. The rapid degradation of the late histone mRNAs was prevented when protein synthesis was inhibited.     

Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.     

Synthesis and turnover of late H2B histone mRNA in developing embryos of the sea urchin, Strongylocentrotus purpuratus

In sea urchins, "early" histone proteins are synthesized during cleavage and blastula formation, "late" histone proteins in subsequent stages of development. To understand the molecular mechanisms responsible for this ontogenic switch in histone subtype synthesis, we determined the absolute amounts, rates of synthesis, and rates of turnover of late H2b histone mRNAs during development. We showed previously that late H2b mRNA comprises several mRNA isotypes. In this study, we used both a class-specific DNA probe to measure the amounts of the late H2b mRNA isotypes collectively, and a gene-specific probe to measure amounts of a particular late H2b mRNA encoded by a gene known as L1. We found that the amount of late H2b mRNA increased dramatically from 85,000 molecules per embryo in the 16-hr blastula to a peak of 670,000 molecules per embryo in the 24-hr mesenchyme blastula, and fell to 380,000 molecules per embryo in the 72-hr pluteus larva. The L1 late H2b mRNA achieved its maximum abundance earlier than the late H2b mRNA class as a whole, reaching a peak of 34% of total late H2b in the 14-hr blastula and declining to 7% in the pluteus larva. Measurements of the rate of incorporation of [3H]uridine into late class H2b mRNA, performed by a novel in vivo isotope incorporation method, enabled us to calculate both synthesis rates and half-lives of late H2b mRNA during development. These calculations showed (1) that the increase in late H2b mRNA level between 16 and 24 hr postfertilization is regulated primarily if not entirely at the level of mRNA synthesis; and (2) that the half-life of late H2b mRNA is comparatively short, around 20 min, at all stages examined.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.