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.     

Length and sequence heterogeneity of the histone gene repeat unit of the sea urchin, S. purpuratus

Histone gene repeats in S. purpuratus are shown to be of variable length and sequence. Two recombinant plasmids containing the full-length 6.3 kb histone repeat unit are found to differer in length at two sites in the repeating structure and in the occurrence of two restriction enzyme recognition sites. Variation in repeat length is also demonstrated in the unfractionated DNA of five sea urchins and in a sample of DNA enriched for histone gene sequences by density gradient methods. The repeats in each individual are of a very limited number of major classes, which may differ from one another in overall length or in distribution and presence of particular restriction enzyme sites. Variations are found to occur at many regions of the repeat; some have been mapped specifically to spacer regions. Repeats may differ dramatically from individual to individual since there is no one type of repeat class common to all, although the absolute length differences of the repeats that are found are small.     

The single-copy DNA sequence polymorphism of the sea urchin strongylocentrotus purpuratus

The single-copy DNA sequence difference between individual sea urchins of the species Strongylocentrotus purpuratus has been estimated by comparing the thermal stability of reassociated DNA duplexes from two individuals with that for DNA from an individual. Thermal stability was measured by hydroxyapatite thermal chromatography, S1 nuclease resistance after heating in a solvent which neutralizes the effect of DNA base composition, and spectrophotometric melting. One pair of individuals appear to differ from each other in about 4% of the nucleotide pairs of their single-copy DNA sequence. The differences in DNA sequence among individuals in local populations are not distinguishably smaller than those among populations as far apart as 2000 kilometers along the Pacific coast of North America.     

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 genes of the sea urchin (S. purpuratus) cloned in E. coli: Order,polarity, and strandedness of the five histone-coding and spacer regions

Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I, Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions.Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNAs have little sequence homology to other genes. Exonuclease III digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5′-3′ polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of the S. purpuratus histone gene repeat unit:

     

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.     

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.     

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, $\text{dP1}\text{dt}\text{= M·f·n·}\text{A}\text{t}$, where $\text{dP1}\text{dt}$ = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and $\text{A}\text{t}$ = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ($\text{dP1}\text{dt}$). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo^?1 hr^?1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo^?1 hr^?1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (f^zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f^zygotic and $\text{dP1}\text{dt}$, the rate of elongation of nascent histone polypeptide chains ($\text{A}\text{t}$) during the first 6 hr of development was estimated; $\text{A}\text{t}$ remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.     

Spectroscopic studies on histone-DNA interactions. I. The interaction of histone (H2A, H2B) dimer with DNA: DNA sequence dependence

Binding of the histone (H2A, H2B) dimer with chicken erythrocyte DNA has been studied by salt-titration spectroscopy in equilibrium conditions. The circular dichroism of DNA near 275 nm is depressed by the interaction with (H2A, H2B) at low concentrations of salt. The depression increases with increasing amounts of (H2A, H2B), and reaches a plateau at an (H2A, H2B) to DNA ratio of 1.5 (w/w), at which one (H2A, H2B) dimer occupies 28 base-pairs of DNA. The fluorescence emission intensity of the tyrosine residues in (H2A, H2B) is depressed by the H2A, H2B)-DNA interaction. When the DNA-(H2A, H2B) complex is titrated with NaCl, these two signals show transitions with increasing ionic strength of the buffer, whose normalized transition curves agree well. The midpoint of the transition is about 0.42 M-NaCl for a sample with a DNA concentration of 0.05 mg/ml and an (H2A, H2B) to DNA ratio of 0.4 (w/w). The fluorescence titration curves have been analyzed to obtain the binding constant for the (H2A, H2B) dimer with DNA. The sample concentration dependence of the titration profiles is consistent with the model of non-cooperative binding of (H2A, H2B) dimer to DNA. The titration profiles are reversible. The obtained binding constant for the (H2A, H2B) dimer with chicken erythrocyte DNA at 20 degrees C (pH 7.6), as a function of the ionic strength, I, is as follows: log10K = -14.9 log10(I)-1.2. The change of enthalpy delta H accompanied by the binding of the (H2A, H2B) dimer is nearly equal to zero, within an error of +/- 1.4 kcal/mol (1 cal = 4.184 J). DNA sequence dependence of the stability of DNA-(H2A, H2B) interactions is observed using reconstituted materials of synthetic DNAs. A decreasing stability of the interaction is observed following the order: the duplex of poly[(dA)-(dT)] greater than chicken erythrocyte DNA or the copolymer duplex of poly(dA).poly(dT) greater than the duplex of poly[(dG)-(dC)]. The difference in free energy of the association of the (H2A,H2B) dimer between the two copolymers is 0.8 kcal/mol.     

Evolving sea urchin histone genes-nucleotide polymorphisms in the H4 gene and spacers ofStrongylocentrotus purpuratus

Summary We present a comparison of spacer and coding sequences of histone gene repeats from fourStronglycocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions: tranversions: single-base-pair insertions/deletions was 322. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)_n and (CT)_n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of everyS. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pariwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1–2 million years necessary for accumulation of the variation. The level of sequence variation found within theS. purpuratus population, for both tandemly repeated and single-copy genes, is 25%–50% of that found betweenS. purpuratus andS. drobachiensis.     

Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

A regulatory sequence near the 3'' end of sea urchin histone genes.

The 3' flanking sequences of all five histone genes have been sequenced in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. A large (23 bp) and a small (10 bp) conserved sequence was found by sequence comparison, some 29-40 bp downstream from the termination codon. 12 bases of the larger homology block show a dyad symmetry. The available sequences of clone h22 of the same species and those of the histone clones pSp2 and pSp17 of Strongylocentrotus purpuratus, another sea urchin species, fit well into this comparison. Two types of sequences are involved in the dyad symmetry; one is H1, H3 and H4 specific, the other is H2A and H2B specific. If these conserved sequences are transcribed, a hairpin loop could form in the RNA molecules. This secondary structure might serve as a recognition signal for a regulatory protein.     

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.     

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 cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.