Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

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.     

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.     

Initial characterization of sulfated macromolecules in the blastocoels of mesenchyme blastulae of Strongylocentrotus purpuratus and Lytechinus pictus

Sulfated macromolecules have been implicated in many morphogenetic activities, including cell migration and gastrulation in echinoid embryos. Since the blastocoel is a major site for the accumulation of sulfated macromolecules, this study focuses on an initial characterization of sulfated components present in isolated blastocoelic matrix from mesenchyme blastula-stage embryos of Strongylocentrotus purpuratus and Lytechinus pictus. Based on transmission and scanning electron microscopy, the isolated matrix resembles that seen in situ, containing 25- to 30-nm-diameter granular and small fibrous components. Only a small proportion of the labeled material enters 7.5% polyacrylamide gels to separate in a number of species-specific bands larger than 30,000 daltons. A major portion of the label is voided from Sepharose CL-2B columns prepared in dissociative solvent. Based on the chromatographic isolation of digestion products after treatment with chondroitinase AC and ABC, in S. purpuratus about 37% of the label is in dermatan sulfate and 16% in chondroitin 6-sulfate. In contrast, in L. pictus about 25% of the label is in dermatan sulfate with only low levels of chondroitin sulfate. In both species, based on nitrous acid and heparinase sensitivity of Pronase digests prior to chromatography on Sephadex G50, about 13% of the label is in heparan sulfate. A small residual fraction remains uncharacterized.     

Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins

Two peptides associated with the eggs of the sea urchin, Lytechinus pictus, which stimulate L. pictus but not Arbacia punctulata sperm respiration rates, were purified and their amino acid sequences determined. The peptides (Gly-Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln and Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln) were found to be structurally similar to the peptide, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly). Chemical synthesis of the two peptides confirmed their ability to activate sperm respiration. The peptides had equivalent biological activity with half-maximal stimulation of respiration rates and of cyclic nucleotide concentrations occurring at 60 pM and 700 pM, respectively. Addition of the peptides to intact spermatozoa resulted in the rapid appearance of a newly-stained protein on Na X dodecyl X SOl polyacrylamide gels (Mr = 140,000); one-half maximal formation of the Mr 140,000 protein occurred at about 20-100 nM peptide.     

Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus

Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly. Phosphorylation is on serine residues in the N-terminal fragment of H1 bisected with N-bromosuccinimide. Phosphorylation is maximal by 4-8 min and dependent on Ca2+, but independent of Na+ or increased intracellular pH. Phosphorylation of H1 can be dissociated from the induction of the acrosome reaction. Only a fraction of the H1 molecules become phosphorylated upon treatment of sperm with egg jelly. The amount of phosphate per mole of H1 increases from 0.15 moles before jelly treatment to 0.46 moles after maximal phosphorylation. Phosphorylation of H1 occurs in a cAMP-dependent manner as indicated by the ability of the phosphodiesterase inhibitors IBMX and SQ20009 to induce H1 phosphorylation. This phosphorylation reaction can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin, showing that a ligand in egg jelly must interact with a sperm surface receptor to activate the kinase phosphorylating H1.     

The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms

Adult tissues of the sea urchin, Strongylocentrotus purpuratus, were analyzed for the products of a set of genes whose expression, in the embryo, is restricted to the skeletogenic primary mesenchyme (PM). Three embryonic PM-specific mRNAs were found to be abundant in adult skeletal tissues (test and lantern), but not in a variety of soft tissues. Homologous mRNAs were also found in skeletal tissues of the congeneric sea urchin, S. droebachiensis, as well as a more distantly related echinoid, Dendraster excentricus, and an asteroid, Evasterias troschellii. The distributions of two of these RNAs were analyzed in regenerating spines of adult S. purpuratus using in situ hybridization. These gene products were localized primarily in the calcoblasts that accumulated at the regeneration site. In nonregenerating spines SpLM 18 RNAs, the most abundant of these gene products, were localized in a small population of noncalcoblast cells scattered through the spine shaft, and were absent from calcoblasts. These observations suggest that a program of gene expression associated with the process of calcification is conserved both developmentally through the period of metamorphosis and evolutionarily among the echinoderms.