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.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Atypical protein kinase C controls sea urchin ciliogenesis

The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.     

Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Summary The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease Sl fromAspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form Sl resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with Sl nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive Sl nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9°C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.Also Staff Member, Carnegie Institution of Washington     

Cyclin E in centrosome duplication and reduplication in sea urchin zygotes

When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.     

Identification of a calsequestrin-like protein from sea urchin eggs

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.     

Genetic divergence and assortative mating between colour morphs of the sea urchin Paracentrotus gaimardi

Some species of sea urchins feature large variation in pigmentation. This variability may be the result of phenotypic plasticity or it may be associated with genetic divergence between morphs. Paracentrotus gaimardi exhibits five colour morphs (pink, brown, green, grey and black), which often occur side by side on the same rock. We studied genetic divergence between these morphs in three populations on the coast of Brazil. A fragment of the region encoding the mitochondrial ATPase 8 and 6 mitochondrial genes, a fragment of the intron of a nuclear histone and the entire nuclear gene coding for the sperm protein bindin were analysed. Mitochondrial DNA was differentiated between the pink and all other morphs, but the histone intron was similar in all colour morphs. In bindin, nine codons were found to be under positive selection and significant differences of allelic frequencies were observed in almost all pairwise comparisons between colour morphs. Although the molecular differentiation in bindin is not large enough to suggest reproductive isolation, some degree of assortative mating within morphs seems to be occurring in this species.     

Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins

Comparison of the nucleoside triphosphate-binding motif(NTBM)-containing proteins of two groups of apparently distantly related positive-strand RNA viruses (potyvirus and flavivirus), revealed significant sequence similarity. In addition, these two groups of viral proteins show amino acid motifs in common with those conserved in a group of five NTBM-containing proteins from prokaryotic and eukaryotic cells, some of which have been experimentally related to helicase activity. Here we propose that the proteins mentioned above constitute a superfamily of helicase-like proteins, distinct from the one previously described [Gorbalenya et al., FEBS Lett. 235 (1988) 16-24; Hodgman, Nature 333 (1988) 22-23; 578], which includes the NTBM-containing proteins from another group of positive-strand RNA viruses, the 'Sindbis-like' viruses.     

A putative precursor to the major yolk protein of the sea urchin

We detected by electrophoresis, several glycoproteins in the eggs of three species of sea urchin. The major glycoprotein band disappears as development of the embryo proceeds. This protein is enriched in the yolk fraction obtained by zone sedimentation in 2.5–30% sucrose gradients. A fractionally larger glycoprotein has been found to be the major protein in the coelomic fluid of male and female gravid sea urchins. Partial proteolysis peptide mapping shows that the major coelomic fluid protein and the major yolk protein are related, presumably in a precursor-product relationship.     

Molecular phylogenies and divergence times of sea urchin species of Strongylocentrotidae,Echinoida

Sea urchins of the family Strongylocentrotidae have been important model systems in many fields of basic biology, yet knowledge of their evolutionary identities such as the phylogenetic relationships and divergence times remains limited. Here, I inferred molecular phylogenies of seven Strongylocentrotid species (Strongylocentrotus franciscanus, S. nudus, S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus, and Hemicentrotus pulcherrimus) from the analyses of mitochondrial DNA sequences of 12SrDNA (349 nt), 12SrDNA-tRNA(gln) region (862 nt), and a combined sequence of cytochrome oxidase subunit I (COI, 1080 nt) and NADH dehydrogenase subunit I (NDI, 742 nt). The rate of sequence evolution and divergence times for each species were then estimated from the trees with reference to the time of separation between Strongylocentrotidae and Parechinidae, 35 to 50 MYA. The three trees agree well with each other, and the phylogeny is summarized by ((S. franciscanus, S. nudus), (H. pulcherrimus (S. purpuratus, S. intermedius (S. droebachiensis, S. pallidus)))). It is notable that the genus Strongylocentrotus consists of two distinct clades and that H. pulcherrimus branches off within Strongylocentrotus, implying assignment of a separate, monospecific genus to this species inappropriate. The rate of sequence evolution is calibrated to be 0.24%-0.34%/Myr in 12SrDNA, 0.25%-0.36%/Myr in 12SrDNA-tRNA(gln), and 0.65%-0.93%/Myr in COI-NDI combined sequences. S. purpuratus, in particular, shows the significantly higher rate of evolution in the 12SrDNA and 12SrDNA-tRNA(gln) regions compared to other species, suggesting careful use of its sequences in comparative studies. The two clades of Strongylocentrotidae seem to have split 13-19 MYA, and H. pulcherrimus branched off 7.2-14 MYA. In the former clade, S. franciscanus and S. nudus separated 5.7-8.1 MYA. In the latter clade, S. purpuratus, S. intermedius, and the clade of S. droebachiensis and S. pallidus diverged approximately 4.6-12 MYA, and the last two closest species separated 2.1-3.1 MYA.     

Resolution and characterization of a major protein of the sea urchin hyaline layer

A major protein component of the gel-like, embryonic hyaline layer of Strongylocentrotus purpuratus has been purified and characterized. The protein retains the ability to form an insoluble gel in the presence of specific divalent cations, a property characteristic of the hyaline material. Using a light scattering assay developed to measure the initial rate of hyalin gelation, we have been able to show that calcium alone is capable of initiating this reaction but that calcium and magnesium are synergistic in their effect. In the absence of divalent cations, the major hyalin protein has a molecular weight of 9.2 +/- 0.5 X 10(5) and a sedimentation coefficient of 11.6 S; these and other data indicate that the protein assumes a very elongated, rod-like structure in solution. Smaller amounts of two additional proteins, 8.8 and 6.5 S, are present in the hyalin fraction when the jelly coat and vitelline layer are subjected to a more stringent acid treatment early in the isolation procedure.     

Characterization of proteins belonging to the CHAP-related superfamily within the Firmicutes

Cell division is a dynamic process ending by separation of the daughter cells. This final step requires the cleavage of the murein septum synthetized during cell division. In Streptococcus thermophilus, cse plays an important role in cell separation. Cse protein contains, at its N-terminal end, a signal peptide and a putative LysM motif suggesting that it is secreted and able to bind to the cell wall. Furthermore, the C-terminus of Cse carries a putative cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain conferring to the protein a potential catalytic activity. To gain insight into the role of Cse in the cell division process, in silico analysis of the Firmicutes proteins displaying CHAP-related domain was undertaken. This work allowed us to distinguish and characterize within the Firmicutes the 2 families of proteins (CHAP and NlpC/p60) belonging to the CHAP superfamily. These 2 families regroup mainly peptidoglycan hydrolases. Data from the literature indicate that NlpC/p60 and CHAP proteins cleave distinct peptidoglycan bonds. Among the enzymes characterized within the Firmicutes, NlpC/p60 proteins are gamma-D-glutamate-meso-diaminopimelate muropeptidase. Instead, CHAP enzymes involved in cell separation are N-acetylmuramoyl-L-alanine amidase and CHAP lysins have endopeptidase activity.     

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.     

Sperm polymorphism within the sea urchin Strongylocentrotus droebachiensis: divergence between Pacific and Atlantic oceans

The rapid evolution of traits related to fertilization such as sperm morphology may be pivotal in the evolution of reproductive barriers and speciation. The sea urchin Strongylocentrotus droebachiensis has a circumarctic distribution and shows substantial genetic subdivision between northeastern Atlantic populations and northwestern Atlantic and Pacific populations. Using transmission electron microscopy, we show here that sperm shape, size, and ultrastructure differ markedly among populations of S. droebachiensis from different oceans and reflect patterns of genetic divergence. Sperm nuclei from northwestern Atlantic and Pacific populations were longer and narrower than those from the northeastern Atlantic. We additionally demonstrate population-level differences in the amount and location of filamentous actin (F-actin) prior to the occurrence of the acrosome reaction. Sperm from Pacific and northwest Atlantic populations differed from that of all other echinoids examined in that intact sperm contains a partly preformed acrosomal process, a structure more closely resembling the acrosomal rod seen in some molluscs. Immunofluorescent studies using anti-bindin antibodies and the F-actin-specific stain phalloidin confirmed these findings. Divergence of reproductive traits such as sperm morphology may be related to divergence in gamete compatibility and genetic divergence, and could represent the first stages of speciation in free-spawning marine invertebrates.     

Genetic divergence between three sea urchin species of the genus Strongylocentrotus from the Sea of Japan

Intraspecific allozymic variation and interspecific genetic divergence were studied in three sea urchin species of the genus Strongylocentrotus (S. intermedius, S. nudus, S. pallidus) from the Sea of Japan. S. pallidus and S. intermedius showed high mean values of expected heterozygosity, H(e)=0.223+/-0.072 (17loci) and H(e)=0.230+/-0.065 (19loci), respectively. This estimate was somewhat lower in S. nudus, H(e)=0.126+/-0.043 (17loci). Estimates of Nei's genetic distance between S. nudus/S. intermedius (D=1.578, 17loci) and S. nudus/S. pallidus (D=1.327, 15loci) were considerably higher than that between S. intermedius/S. pallidus (D=0.269, 17loci). Invoking the protein clock hypothesis and using Panamanian geminate sea urchins for protein clock calibration, the time of divergence between S. intermedius and S. pallidus was estimated as 2.7MY. The results obtained for S. intermedius and S. nudus by us differ considerably from results obtained for these species by Norimasa Matsuoka and coworkers. The revealed discrepancies are discussed and the conclusion made that Matsuoka and coworkers' data on echinoderm biochemical genetics and systematics should be used with caution.     

Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily

OsIpk and HvIpk, inositol phosphate kinases, were cloned from rice (Oryza sativa L. var. indica, IR64) and barley (Hordeum vulgare) respectively. Sequence alignment showed that they belong to the ATP-grasp family, which includes inositol 1,3,4-trisphosphate 5/6-kinase from humans and Arabidopsis. Residues that are binding sites for ATP and coordinate magnesium in absence or presence of inositol phosphate are conserved and in total 23 residues are invariant among the twelve aligned inositol phosphate kinases. The genes were heterologously expressed in Escherichia coli and kinase activity assays with 17 different isomers of inositol mono-/di-/tri-/tetra-/pentaphosphate as well as phytate were performed. The strongest activity for both kinases was observed with Ins(3,4,5,6)P(4), which candidates as the primary substrate for these kinases in plants. Several species-specific differences between the two recombinant Ipks were observed. Rice OsIpk showed detectable kinase activity towards eight different substrates, whereas barley HvIpk showed kinase activity with all the substrates including inositol mono- and bisphosphates. HvIpk showed 3-kinase activity towards the Ins(1,4,5)P(3) substrate and it also interconverted the two substrates Ins(1,3,4,5)P(4) and Ins(1,3,4,6)P(4) by isomerase activity, which was not observed for the rice homologue. Both OsIpk and HvIpk had no detectable 2-kinase activity. Furthermore, the two Ipks showed phosphatase activity towards several inositol phosphates. Expression analysis by RT-PCR demonstrated that the Ipk gene was equally expressed in different tissues and developmental stages. Taken together, these results show that the Ipk kinase plays a significant role in the inositol phosphate interacting network in plants.