Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

Chromatin-associated proteins of the developing sea urchin embryo. II. Acid-soluble proteins

Of the five well-characterized histories, only the slightly lysine-rich histories F2a and F2b are present in sea urchin embryos before the 16-cell stage. At the 16-cell stage, the arginine-rich (F3) and lysine-rich (Fla) histones appear and all the major histones are then present in the same relative proportions until the pluteus stage except for a second lysine-rich protein, Flb, which is first detected at 12 to 16 hours of development and increases to the pluteus stage. From 16 cells to pluteus at 70 hours, all the histones are labeled by a 30-minute incubation with radioactive lysine, with the exception of the lysine-rich histone Fla which does not incorporate label after 20 to 30 hours of development and Fib which is labeled only after 20 to 30 hours. Fla is conserved, however, to the pluteus stage.The total acid-soluble protein content of chromatin remains constant to 22 hours of development. During the period of 22 to 45 hours, there is a slight loss of protein followed by a rapid loss from 45 to 70 hours such that at 70 hours only 20% remains.     

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.     

Stage Specific Effects on Sea Urchin Embryogenesis of Zn2+, Li+, several Inhibitors of cAMP-Phosphodiesterase and Inhibitors of Protein Synthesis

Treatment of sea urchin embryos for 3 hr starting at the 16-64 cell stage with Li⁺ or 3-isobutyl-1-methylxanthine as well as with other inhibitors of cAMP-phosphodiesterase (PDE) and several inhibitors of protein synthesis, resulted in production of vegetalized embryos with a large exogut. However, the same treatment starting at other stages produced hardly any vegetalized embryos. The specific stage for these substances to cause vegetalization is probably the 16-64 cell stage. Treatment with Zn²⁺ between the times of fertilization and hatching, followed by culture in normal sea water produced animalized embryos with little if any archenteron, but the same treatment followed by culture with ethylenediamine-N, N'-diacetic acid (EDDA), a chelator of Zn²⁺, produced quasi-normal plutei. This chelator did not counteract the animalizing effect of Zn²⁺ when culture with EDDA was started at the post-gastrula stage. Treatment of embryos for a long period (1-3 days) starting at the blastula stage with Li⁺ and the inhibitors of PDE and protein synthesis, as well as with Zn²⁺, produced spherical embryos with little or no archenteron. The stages at which these substances produced abnormal embryos with a poor archenteron are post-hatching stages.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

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.     

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post-hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post-hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3-Aminobenzamide (3-ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP-ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3-ABA in the pre-hatching period had little inhibitory effect on the morphogenesis in the post-hatching period, though it caused death of many embryos.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

Changes in subfraction composition of chicken lysine-rich histone during ontogenesis]

Lysine-rich histone isolated from different chicken tissues was separated electrophoretically into 4-5 subfractions. The subfrations reffered to as 1, 2, 3, and 4, occur in each the tissue studied, erythrocyte lysine-rich histone containing an additional subfraction 1a. F1 histone from mitotically active tissues (intestinal mucosa, thymus, testes) has a higher content of subfraction 2, while the same histones from mototically inactive tissues (liver, heart, brain) contain an elevated amount of subfraction 3. F1 histone isolated from liver, brain and heart of 21-day embryo has much more of subfraction 2, than the same histone of adult animal. During the chicken development from hatching till maturation the content of subfraction 2 in these organs decreases, and the content of subfraction 3 increases. The rate of this change in liver corresponds to the rate of DNA synthesis. In F1 histone of erythrocytes the content of subfraction 4 falls down during the post hatching ontogenesis.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

Synthesis of Proteins Enriched in Li+- Induced Vegetalized Embryos of Sea Urchin during Early Development

Sea urchin embryos were vegetalized by a pulse treatment with 60 mM Li⁺ between 2.5 hr and 6 hr after fertilization at 20°C. Normal and Li⁺ -treated embryos were exposed to [³⁵S]-methionine for 2 hr at various stages and [³⁵S]-labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). On fluorograph of 2D-PAGE at the pre-hatching blastula stage, significant difference of labeled proteins between normal and vegetalized embryos was not observed in the range from neutral to acidic pH, but pls of several proteins were found to be shifted toward alkaline pH. At the mesechyme blastula stage, five major proteins [M.W. 36 K, 43 K (two species), 71 K and 150 K] were enriched in Li⁺-treated embryos among a few hundreds of synthesized proteins. At the late gastrula stage, the labeling intensities of these proteins except for one of 43 K proteins increased remarkably in Li⁺-treated embryos. Furthermore, two proteins (M.W. 105 K and 135 K) were also enriched in Li⁺-treated embryos at this stage. At the prism stage, these proteins enriched in Li⁺-treated embryos became hardly detectable, and the synthesis of at least four proteins (M.W. about 20 K, 41 K, 43 K and 200 K<) appeared to increase in normal embryos, but not in Li⁺-treated embryos. Synthesis of proteins eniched in Li⁺-treated embryos probably support endodermal cell differentiation.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Developmental Patterns in Histones and Histone Synthesis During Early Embryogenesis of the Sea Urchin Paracentrotus lividus

Abstract. Striking developmental changes in histone and histone synthesis in sea urchin embryos were observed in three histone classes, H1, H2A and H2B. In each case there is a shift in histone synthesis from the early cleavage stage types to other types of histones at the morula stage; Two new forms appear after the blastula stage. In addition, multiple changes in histone types were found during gameto-genesis in the male and female gonads where specific histones, different from the embryonic histones, were observed.     

Histones and histone synthesis in sea urchin development

Histones are synthesized and become a part of the chromatin as early as the first cleavage in sea urchins. Reproducible changes in relative amounts of individual histone fractions synthesized are observed during development. A new and electrophoretically distinct very lysine rich fraction appears at hatching in Arbacia and in the early gastrula of Lytechinus. When RNA synthesis is blocked by actinomycin D, maternal mRNA alone can direct a quantitatively and qualitatively changing pattern of histone synthesis as cleavage proceeds. Inhibition of DNA synthesis by hydroxyurea reduces synthesis of histones; the arginine-rich histones are more severely affected than the lysine-rich ones.     

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.