Functional analysis of the promoter of a sea urchin metallothionein gene.

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.     

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.     

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 microg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin-hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.     

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.     

Isolation and characterization of developmentally regulated sea urchin U2 snRNA genes

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.     

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.     

Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos

The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.     

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.     

Biogenesis of the mitochondrial ATPase from sea urchin embryos

The mitochondrial rutamycin-sensitive ATPase from sea urchin eggs was purified to homogeneity. The subunit structure of the enzyme was characterized by SDS-gel electrophoresis. Eight polypeptides were identified with molecular weights of 55,000, 52,000, 39,000, 31,000, 28,000, 23,000, 17,000 and 10,000. Developing sea urchin embryos were incubated with [2H]leucine in the presence of emetine preferentially to label mitochondrially made proteins. Under these conditions sea urchin mitochondria synthesize eight different polypeptides. Two of these proteins, with molecular weights of 31,000 and 23,000, co-purify with the ATPase. Antibody directed against the pure rutamycin-sensitive ATPase precipitated only these two proteins. Therefore, two of the eight sea urchin ATPase subunits appear to be made by mitochondria.     

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 x 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.