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.     

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.     

Action of serotonin antagonists on cytoplasmic calcium levels in early embryos of sea urchin Lytechinus pictus

Possible interaction of the serotonergic system with intracellular calcium mechanisms was investigated using techniques of ratio imaging measurement of intracellular Ca2+ and confocal microscopy in cleaving embryos of sea urchin Lytechinus pictus. Some serotonin antagonists specifically increase free intracellular Ca2+ and evoke transient regression of the first cleavage furrow, suggesting possible linkage of serotonergic and calcium mechanisms in the regulation of cellular events during cleavage divisions. These effects were more pronounced in the experiments with hydrophilic 5-HT-antagonists, quarternary ammonium salts that do not penetrate the cell membrane. Thus, it appears that 5-HT-receptors which mediate these effects are localised on the cell membrane, whereas previously studied receptors mediating the cytostatic action of lipophilic 5-HT-antagonists are localised intracellularly.     

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.     

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.     

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.     

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.     

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.     

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.     

Sequence comparisons of non-allelic late histone genes and their early stage counterparts. Evidence for gene conversion within the sea urchin late stage gene family

We have determined the nucleotide sequence of sea urchin (Lytechinus pictus) late stage H3 and H4 histone genes contained on the clone pLpH3H4 -21 and of the early stage H3 gene contained on the plasmid pLpA . Comparison of these differentially regulated histone genes with each other and with other L. pictus late and early stage histone H3 and H4 genes previously sequenced confirms that members of each histone gene family (early and late) are more homologous to each other than they are to members of other histone gene families. The spacer regions between two late H3-H4 gene pairs on the clones pLpH3H4 -19 and pLpH3H4 -21 have diverged to the point where they are no longer homologous. However, comparative analysis of the 5' flanking DNA has identified a sequence 5'C-T-C-A-T-G-T-A-T-T3' upstream of both late H4 genes and another, 5'A-G-A-T-T-C-A3', upstream of both H3 genes. Except for a short conserved sequence near the initiation codon, the transcribed 5' leaders of the late mRNAs differ in length and sequence in the two non-allelic late histone gene pairs. This divergence contrasts with the 95 to 96% conservation found between late histone gene coding sequences. The results suggest that there is intergenic exchange in the germline among members of the late histone gene family and that the unit of exchange is the individual gene rather than the heterotypic dimer which includes the common spacer DNA.     

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.     

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.     

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.