Proto-oncogene homologous sequences in the sea urchin genome

Using probes specific for several oncogenes/proto-oncogenes we have performed gel blot hybridization analyses of genomic DNA isolated from the sea urchinStrongylocentrotus droebachiensis. Probes prepared from v-erbB, v-myc, c-myb and v-fps were found to hybridize with discrete fragments of HindIII digested genomic DNA. In contrast, probes prepared from v-abl, v-fos, v-sis, v-src, and v-mos either hybridized with multiple fragments, indicating non-specific binding, or failed to hybridize at all above background levels. These results clearly demonstrate the presence of proto-oncogene homologous sequences in the sea urchin genome.     

Calcium–protein interactions in the extracellular environment: Calcium binding,activation, and immunolocalization of a collagenase/gelatinase activity expressed in the sea urchin embryo

We have purified and characterized a collagenase/gelatinase activity expressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymography revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect could be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred with an apparent kd value of 3.7 mM. Two modes of calcium binding to the 41-kDa subunit were detected; up to 80 moles of calcium bound with a kd value of 0.5 mM, while an additional 120 moles bound with a kd value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mol, indicating the availability of substantial numbers of weak Ca²⁺-binding sites. Calcium binding did not result in either secondary or quaternary structural changes in the collagenase/gelatinase, suggesting that Ca²⁺ may facilitate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in blastocoelic fluid and in the hyalin fraction dissociated from 1-h-old embryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed throughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina, respectively. J. Cell. Biochem. 71:546–558, 1998. © 1998 Wiley-Liss, Inc.     

Genes of the sea urchin embryo: An annotated list as of December 1994

The main literature regarding gene structure and expression in sea urchin embryos is schematically reported and briefly commented upon. Although the subject has expanded particularly over the last 10 years, to which the review mostly refers, some historical reference is also given. More space is reserved to the regulation of the synthesis of histones and cytoskeletal actins, where the attention of various authors has been especially present; the regulation of such a synthesis is described both at a territorial level and a temporal level during the sea urchin development.     

Effect of ultraviolet radiation on growth and percent settlement of larval Lytechinus variegatus (Echinodermata: Echinoidea)

Lytechinus variegatus larvae were used to examine the effects of ecologically relevant short-term exposures of ultraviolet radiation (UVR) on larval morphology and settlement. A laboratory experiment tested the blastula, gastrula, and early pluteus stages of larval development for susceptibility to damage from exposure to UVR produced by an artificial light source. Larval post-oral arm length and percent settlement were measured to assess UVR effects. Larvae exposed at the blastula and gastrula stages had a reduction in larval post-oral arm length compared with non-exposure controls, while larvae exposed at the early pluteus stage were similar to controls. Larvae exposed in the gastrula stage had a significant reduction in percent settlement compared with controls. Negative consequences from ecologically relevant levels of UVR were differentially dependent on the larval stage at time of exposure suggesting time point in larval development at exposure will be important in interpreting effects on higher levels of biological organization.     

Adenosine 3′,5′-cyclic monophosphate-binding protein in sea urchin eggs and embryos

Adenosine 3′,5′-cyclic monophosphate (cAMP) binds to high-molecular substances which are probably proteins, in homogenates of sea urchin eggs and embryos. The bound cAMP is exchangeable. Optimal pH for the binding capacity of the proteins with cAMP is 4.0, and is shifted to 5.0 in the presence of 5 mM caffeine. The level of bound cAMP increases steeply during 10 minutes of incubation. This is then followed by a less steep increase. The level of bound cAMP decreases in the presence of NaCl. The dissociation onstant between cAMP and the proteins in homogenates of unfertilized and ertilized eggs is about 10 nM, and the value in the embryos at the gastrula stage is lower than that in the unfertilized egg homogenate.     

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin–neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario—the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are ‘missing links’ in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).     

Changes in cAMP-dependent protein kinase activity in the 10,000g sediment of sea urchin embryos during early development

cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.     

The evolution of the patterning of human lactation: A comparative perspective

Some four decades ago, Jeanne Altmann started her detailed field studies of baboon mothers and their infants with a focus on the behavioral ecology of maternal reproductive investment. 1 Around the same time, Sarah Hrdy studied langur mothers and their infants, focusing on the influence of the social environment on a female's reproductive options and decisions. 2 Their pioneering work has inspired many subsequent studies of female primate reproduction in its natural context and shaped our own work on primate mothers.     

Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin

Huntingtin is a completely soluble 3,144 amino acid (aa) proteincharacterized by the presence of an amino-terminal polymorphicpolyglutamine (polyQ) tract, whose aberrant expansion causesthe progressively neurodegenerative Huntington's disease (HD).Biological evidence indicates that huntingtin (htt) is beneficialto cells (particularly to brain neurons) and that loss of itsneuronal function may contribute to HD. The exact protein domainsinvolved in its neuroprotective function are unknown. Evolutionaryanalyses of htt primary aa have so far been limited to a fewspecies, but its thorough assessment may help to clarify thefunctions emerging during evolution. We made an extensive comparativeanalysis of the available htt protein homologues from differentorganisms along the metazoan phylogenetic tree and defined thepresence of 3 different conservative blocks corresponding tohuman htt aa 1–386 (htt1), 683–1,586 (htt2), and2,437–3,078 (htt3), in which HEAT (Huntingtin, Elongatorfactor3, the regulatory A subunit of protein phosphatase 2A,and TOR1) repeats are well conserved. We also describe the cloningand sequencing of sea urchin htt mRNA, the oldest deuterostomehomologue so far available. Multiple alignment shows the firstappearance of a primitive polyQ in sea urchin, which predatesan ancestral polyQ sequence in a nonchordate environment anddefines the polyQ characteristic as being typical of the deuterostomebranch. The fact that glutamines have conserved positions indeuterostomes and the polyQ size increases during evolutionsuggests that the protein has a possibly Q-dependent role. Finally,we report an evident relaxing constraint of the N-terminal blockin Ciona and drosophilids that correlates with the absence ofpolyQ and which may indicate that the N-terminal portion ofhtt has evolved different functions in Ciona and protostomes.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.