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.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

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.     

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.     

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.     

Putative nuclease-sensitive control element in unfertilized eggs of the sea urchin Lytechinus pictus

Very limited nuclease-treatment of actively translating lysates from unfertilized eggs of the sea urchin Lytechinus pictus results in activation of the lysate. This activation is reflected by the ability of the nuclease-treated (or 'nuclease-activated') lysates to more efficiently utilize exogenous added RNAs. The activation may be reversed by adding small aliquots of untreated lysate to the nuclease-activated lysates. These results suggest that a nuclease-sensitive negative control element may be involved in the translational repression of unfertilized eggs, and that a deactivation of this control element is partially responsible for the dramatic activation of protein synthesis following fertilization in sea urchin eggs.     

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.     

NO/cGMP signaling and HSP90 activity represses metamorphosis in the sea urchin Lytechinus pictus.

Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod. We present evidence for a similar role in the sea urchin Lytechinus pictus. NO commonly signals via soluble guanylyl cyclase (sGC). Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in echinoid larvae as well. Pluteus larvae containing juvenile rudiments were treated with either radicicol L- or D-nitroarginine-methyl-ester (L-NAME and D-NAME), or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitors of HSP90, NOS, and sGC, respectively. In all instances, drug treatment significantly increased the frequency of metamorphosis. SNAP, a NO donor, suppressed the inductive properties of L-NAME and biofilm, a natural inducer of metamorphosis. NADPH diaphorase histochemistry indicated NOS activity in cells in the lower lip of the larval mouth, the preoral hood, the gut, and in the tube feet of the echinus rudiment. Histochemical staining coincided with NOS immunostaining. Microsurgical removal of the oral hood or the pre-oral hood did not induce metamorphosis, but larvae lacking these structures retained the capacity to metamorphose in response to ODQ. We propose that the production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduces the production of NO, and consequently cGMP, to initiate metamorphosis.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

Organization of the ciliary basal apparatus in embryonic cells of the sea urchin,Lytechinus pictus

Summary The basal apparatus of embryonic cells of the sea urchin Lytechinus pictus was examined by transmission electron microscopy and compared with the basal apparatus of other metazoan cells. The basal apparatus in these cells is associated with a specialized region of the apical cell surface that is encircled by a ring of microvilli. The basal apparatus includes several features that are common to all ciliated cells, including a basal body, basal foot, basal foot cap, and striated rootlet. However, a component not seen in the basal apparatus of other species has been observed in these cells. This structure is continuous with the striated rootlet, and its ultrastructure indicates that it is composed of the same components as the rootlet. This structure extends from the junction of the basal body and striated rootlet to the cortical region that surrounds the basal body. Based on its morphology and position, this structure is referred to as a striated side-arm. The striated side-arm is always aligned in the plane of the basal foot. Thus, both of these structures extend from the basal body in the plane of the effective stroke. It is suggested that the striated side-arm serves to stabilize the basal apparatus against force exerted by the cilium.     

A new family of tandem repetitive early histone genes in the sea urchin Lytechinus pictus: evidence for concerted evolution within tandem arrays.

We have isolated and characterized a third nonallelic tandemly arrayed histone cluster (LpE) from the sea urchin Lytechinus pictus. Although this tandem array is not intermingled with the other two early histone gene families also found in the L. pictus genome, the order and polarity of the five histone coding sequences in this family are the same as every other well characterized sea urchin early histone gene family. Heteroduplex analysis and restriction endonuclease mapping experiments indicate that the LpE family is more closely related to the B-C than the A-D family of early histone genes. Examination of several individual sperm DNA samples has revealed considerable polymorphism in each of the three tandem repeat families. Within an individual, however, each family is remarkably homogeneous. Thus, our results indicate that rapid fixation of variants acts to homogenize the members of a single tandem array at a considerably faster rate within a family than between families. However, at least some exchange of sequences between families is evident based on the conservation of many restriction endonuclease recognition sites and from analysis of a a cosmid clone in which the A-D and E tandem repeats are found adjacent to one another. These differences in the rate of fixation of variants within and between these families are likely to be responsible for the maintenance of diversity between the different families.     

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.     

A developmentally regulated α2,8-polysialyltransferase in embryos of the sea urchin Lytechinus pictus

The recent chemical identification of polysialylated glycoproteins in the jelly coat and on the cell surface of the sea urchin egg raises important questions about their biosynthesis and possible function. Using CMP-[¹⁴C]-Neu5Ac as substrate and cell free preparations from eggs and embryos of the sea urchin Lytechinus pictus , we have identified a membrane associated CMP-Neu5Ac:poly-α2,8 sialosyl sialyltransferase (polyST) that transferred Neu5Ac from CMP-Neu5Ac to an endogenous acceptor membrane protein of approximately 38kDa. An average of five to six [¹⁴C]-Neu5Ac residues were transferred to the glycan moiety of this protein. The membrane-associated polyST also catalyzed the polysialylation of several exogenous mammalian ganglioside acceptors, including G_D3. Given that no structurally similar naturally occurring polysialylated gangliosides have been described, nor were observed in the present study, we conclude that a single polyST activity catalyzes sialylation of the endogenous acceptor protein and the gangliosides. Using an excess of G_D3 as an exogenous acceptor, it was established that the expression of the polyST in L. pictus embryos increased rapidly at the mesenchyme blastula stage and reached a maximum at the gastrula stage. The finding that this polyST in the sea urchin embryo is developmentally regulated raises the possibility that it may play a role in the changing cell and tissue interactions that occur during gastrulation and the early stages of spicule formation.