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 characterization of spicule proteins from Strongylocentrotus purpuratus

A simple method is described for the isolation of spicules from pluteus embryos of the sea urchin, Strongylocentrotus purpuratus. Radio-iodination of the demineralized matrix reveals six bands on SDS protein gels. Treatment with N-glycanase leads us to believe that some of these proteins are N-linked glycoproteins.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

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.     

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

Microsatellite loci in wild-type and inbred Strongylocentrotus purpuratus

Strongylocentrotus purpuratus, a major research model in developmental molecular biology, has been inbred through six generations of sibling matings. Though viability initially decreased, as described earlier, the inbred line now consists of healthy, fertile animals. These are intended to serve as a genomic resource in which the level of polymorphism is decreased with respect to wild S. purpuratus. To genotype the inbred animals eight simple sequence genomic repeats were isolated, in context, and PCR primers were generated against the flanking single-copy sequences. Distribution and polymorphism of these regions of the genome were studied in the genomes of 27 wild individuals and in a sample of the inbred animals at F2 and F3 generations. All eight regions were polymorphic, though to different extents, and their homozygosity was increased by inbreeding as expected. The eight markers suffice to identify unambiguously the cellular DNA of any wild or F3 S. purpuratus individual.     

The genomic underpinnings of apoptosis in Strongylocentrotus purpuratus

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a death domain, suggesting that echinoderms have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of caspases. While the set of effector caspases (caspases-3/7 and caspase-6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone echinoderm-specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the vertebrate interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of vertebrates, and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in animals and was subsequently lost in the nematode and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or nematodes, but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism.     

Purification and partial characterization of hatching protease of the sea urchin, Strongylocentrotus purpuratus.

The supernatant above hatched sea urchin (Strongylocentrotus purpuratus) blastulae contains crude hatching protease, which is heterogeneous in molecular weight, solubility, charge, and density. It requires urea treatment (6 m, 22 °C, 6 h) to dissociate from the enzyme the heterogeneous population of fragments it has generated in digesting its substrate, the fertilization envelope. It can then be purified 340-fold by diethylaminoethyl-cellulose, ammonium sulfate, and Sephadex G-100. The resulting preparation, homogeneous by the criteria of gel exclusion chromatography, sodium dodecyl sulfate gel electrophoresis, and thermal inactivation, has the following properties: specific activity = 1.44 U mg^?1 (1.44 μmol min^?1 mg^?1); k_cat = 0.72 s^-1; molecular weight = 29,000; energy of activation = 12.9 kcal mol^?1 on dimethylated casein;K_m = 0.93 mgml^?1 dimethylated casein. The pure enzyme is optimally active at pH 7 to 9, 0.5 m NaCl, 10 mm Ca²⁺, and 42 °C. Purification renders the enzyme less stable to freezing and thawing and increases the rate of its thermal inactivation at 37 °C by 100-fold.     

Histone mRNA in eggs and embryos of Strongylocentrotus purpuratus

Histone messenger RNA is detectable in both the maternal RNA which is stored in the unfertilized sea urchin egg and in the RNA species which are synthesized $\text{de}$$\text{novo}$ after fertilization. Hybridization competition experiments show that sequences similar to pulse-labeled 912S RNA from morulae are present in total RNA from unfertilized eggs as well as that from later stages. The proportion of histone mRNA in cellular RNA increases after fertilization, reaching a maximum at the morula stage. Although these messengers are still present in hatched blastulae and gastrulae, they represent a smaller proportion of total RNA compared with earlier stages.     

Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.