Evolution of late H2A, H2B, and H4 histone genes of the sea urchin, Strongylocentrotus purpuratus.

Sea urchins possess several distinct sets of histone genes, including "early" genes, maximally active in cleavage and blastula stages, and "late" genes, active from the late blastula stage onwards. We determined the nucleotide sequences of six sea urchin (Strongylocentrotus purpuratus) late histone genes located on four genomic segments. Comparative analysis of these sequences identified several conserved elements in 5' flanking regions, including the sequences ATGPyATANTATA shared by all late genes and GGCGGGAAATTGAAAA shared by two late H4s. Comparisons of protein-coding sequences of late H4 and H2B genes with their early counterparts showed that silent sites have diverged to the theoretical maximum, indicating that early and late histone gene classes diverged at least 200 million years ago. Since extant echinoderms evolved from a common ancestor at about that time, it is likely that early and late histone gene sets are characteristic of all echinoderm groups. Amino acid sequences derived from nucleotide sequences of late H2A and H2B gistone genes differ substantially from amino acid sequences of their late counterparts. Most such differences are in highly mutable positions. A few, however, occur in positions that do not mutate frequently and thus may reflect functional differences between the early and late forms of the H2A and H2B proteins.     

A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.     

Phosphorylation of sea urchin histone CS H2A

Phosphorylation of cleavage stage (CS) histones was studied during the first cell cycle in male pronuclei of the sea urchin. Histone CS H2A rapidly incorporated 32PO4 during the replication period, but not before. Peptide mapping and amino acid analysis of radiolabelled CS H2A showed that phosphorylation occurred mainly on serine residues located in the C-terminal region of the molecule. When DNA replication was inhibited with aphidicolin both CS H2A and CS H2B accumulated in male pronuclei at the same rate as in the control culture, whereas accumulation of H3 and H4 histones was reduced. Incorporation of 32PO4 by CS H2A doubled when DNA synthesis was inhibited with aphidicolin. Thus phosphorylation of CS H2A was correlated with transport of CS histones from the egg storage pool to the male pronucleus, but not with chromatin synthesis, indicating that this event precedes nucleosome formation. A role for phosphorylation and dephosphorylation of the CS H2A C-terminal region in modulating transport of stored CS histone dimers and their assembly into nucleosomes is discussed.     

Characterization and expression of two matrix metalloproteinase genes during sea urchin development

Matrix metalloproteinases (MMPs) play an essential role in a variety of processes in development that require extracellular matrix remodeling and degradation. In this study, we characterize two MMPs from the sea urchin Strongylocentrotus purpuratus. These clones can both be identified as MMPs based on the presence of conserved domains such as the cysteine switch, zinc-binding, and hemopexin domains. In addition, both of these genes contain consensus furin cleavage sites and putative transmembrane domains, classifying them as membrane-type MMPs. We have named these clones SpMMP14 and SpMMP16 based on the vertebrate MMPs with which they share the greatest similarity. SpMMP14 is expressed in all cells from the egg to mesenchyme blastula stage embryo. Expression of this gene is strongest in the animal and vegetal poles early in gastrulation and in the animal pole only later in gastrulation. SpMMP16 is expressed at low levels in eggs. Expression of SpMMP16 becomes more pronounced in the vegetal pole region at the blastula and mesenchyme blastula stages and becomes confined to vegetal pole descendants, such as pigment cells, later in development. In the future, we hope to learn more about the possible functions of these genes in sea urchin development.     

Stage-specific mRNAs coding for subtypes of H2A and H2B histones in the sea urchin embryo

Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties.Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is Impaired from early prophase through early G1, whereas phagocytosis of non-opsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells.Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37°C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphase.The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization.It is evident that membrane processes which depend upon endocytosis-for example, certain hormone-induced signals-may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.     

Characterization of a metalloproteinase: A late stage specific gelatinase activity in the sea urchin embryo

We have partially purified and characterized an 87 kDa gelatinase activity expressed in later stage sea urchin embryos. Cleavage activity was specific for gelatin and no cleavage of sea urchin peristome type I collagen, bovine serum albumin or casein was detected. Magnesium and Zn²⁺ inhibited the gelatinase and Ca²⁺ protected against inhibition. Ethylenediamine tetracetic acid, ethylenebisoxyethylenenitriol tetraacetic acid and 1,10-phenanthroline were inhibitory, suggesting that the gelatinase is a Ca²⁺- and Zn²⁺-dependent metalloproteinase. No inhibition was detected with serine or cysteine protease inhibitors and the vertebrate matrix metalloproteinase (MMP) inhibitor, Batimastat, was also ineffective. The vertebrate MMP activator p-aminophenylmercuric acetate was without effect. These results allow us to identify both similarities and differences between echinoderm and vertebrate gelatinases. J. Cell. Biochem. 66: 337–345, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Distribution of histone H1 alpha among cells of the sea urchin embryo

We have used immunofluorescent staining of sea urchin embryos to study how histone H1 alpha is distributed among progeny cells formed after the cessation of its synthesis. Our results are consistent with H1 alpha being distributed to both daughter cells at mitosis, resulting in it being most concentrated in cells that stop dividing shortly after H1 alpha synthesis ends, while cells that continue to divide dilute their H1 alpha content in proportion to the number of cell divisions. This rules out our earlier suggestion that H1 alpha becomes segregated in dividing cells. In addition, our results show that most dividing cells of the 3-day embryo contain predominantly H1 beta and H1 gamma. Since these subtypes are known not to undergo phosphorylation, this finding has implications regarding the roles of H1 phosphorylation in the cell cycle.     

Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.