Mitochondrial DNA in the sea urchin Arbacia lixula: nucleotide sequence differences between two polymorphic molecules indicate asymmetry of mutations.

Two polymorphic forms of mitochondrial DNA (mtDNA) extracted from Arbacia lixula eggs were cloned and the nucleotide sequences of specific regions determined. A comparison of the sequences of the sense strand of the two molecules demonstrates that all the differences are transitions and only of the A----G type. A change such as G----A (or A----G) on the sense mtDNA strand results from either a direct G----A (or A----G) mutation on that strand or a C----T (or T----C) on the complementary strand. None of the C----T (or T----C) changes were detected on the sense strand, which implies that the A----G mutation bias on the sense strand is not reversed for the other strand. Our observation indicates the existence of mechanisms acting asymmetrically on the two mtDNA strands, possibly during mtDNA replication.     

Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.      

Intermediary metabolism in sea urchin: the first inferences from the genome sequence

The genome sequence of the purple sea urchin Strongylocentrotus purpuratus recently became available. We report the results of functional annotation and initial analysis of more than 2300 proteins predicted to be involved in metabolite transport and enzymatic conversion in sea urchin. The comparison of various reconstructed biosynthetic and catabolic pathways in sea urchin to those known in other genomes suggests the overall similarity of the sea urchin metabolism to that of the vertebrates, with relatively small but non-trivial differences from both vertebrates and protostomes. There are several examples of two parallel, non-orthologous solutions for the same molecular function in sea urchin, in contrast with the other completely sequenced metazoans that tend to contain just one version of the same function. There are also genes that appear to be close phylogenetic neighbors of plant or bacterial homologs, as opposed to homologs in other Metazoa. The evolutionary and functional significance of these variations is discussed.     

Reception and extrareceptor binding of cytostatic serotonin antagonists by early embryos of the sea urchin Arbacia lixula

Unfertilized eggs and early embryos of the sea urchin Arbacia lixula incubated for 60 min in a medium containing the antagonists of prenervous serotonin, i.e. inmecarb (21 microM) or imipramine (40 microM), bind up to 5 microM of these drugs per 1 ml of cells. At high cell concentrations (more than 10,000 eggs or embryos per 1 ml), this binding is not followed by inhibition of cleavage divisions or by increase in the sensitivity to cytostatic effects of these drugs, which is taken as an indication that this binding is a nonreceptive one. The decrease in concentration of eggs or embryos does not affect total binding of the drugs, although their antiserotonin effects become evident indicating the existence of the receptor sites of binding. In experiments with 3H-imipramine, two binding pools were found (Bmax being correspondingly equal to about 20 and 0.75 microM/ml of embryos; the values of Kd amount to 200 and 15 microM). One of them is a nonreceptive pool, whereas the other presumably coincides with receptor binding sites of prenervous serotonin antagonists.     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Species-specific dissociation into single cells of live sea urchin embryos by fab against membrane components of Paracentrotus lividus and Arbacia lixula

The species and stage specificities of membrane components active in promoting reaggregation of cells dissociated from embryos of the two Mediterranean sea urchin species Paracentrotus lividus and Arbacia lixula have been examined. Membrane proteins extracted with butanol either from purified membranes or from dissociated cells without significant reduction of viability promoted reaggregation of both the homologous and heterologous species. Extracts from plutei and blastulae were equally effective in promoting reaggregation of blastula cells. By contrast, Fab's prepared from IgG raised against these extracts or purified membranes are strictly species specific because they prevent reaggregation of cells and actively dissociate live embryos of only the homologous species. No corresponding stage specificity of the Fab was observed: Fab against extracts from blastula embryos also caused dissociation of plutei. Antigenic analysis of the extracts by the Ouchterlony test revealed the presence of components specific for each species as well as others common to both.     

Two-dimensional electrophoretic analysis of major phosphoproteins of the sea urchin, Arbacia punctulata

Phosphoproteins of hatched blastulae, gastrulae, and pluteus larvae of the sea urchin, Arbacia punctulata, were labeled in vivo with [32P]O4 and analysed by 2-dimensional polyacrylamide gel electrophoresis and autoradiography. At least 60 phosphoproteins were resolved. Some of these showed different relative intensities of labeling at the embryonic periods monitored. Some embryonic phosphoproteins were characterized by cell fractionation and by comparing autoradiograms with Coomassie-blue staining patterns and [35S]methionine labeling patterns. Neither actin nor tubulin phosphorylation was detected. No differences in phosphorylation were detected in dissociated and partially reassociated blastula cells relative to each other and to intact embryonic controls.     

Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.     

Temperature induced isozyme variants in individuals of the sea urchin, Arbacia punctulata.

1. The technique of microacrylamide slab gel electrophoresis was used to repeatedly monitor the qualitative isozyme composition in the tube feet of the sea urchin, Arbacia punctulata exposed to different temperature regimes. 2. Four enzyme systems were assayed. Three (MDH, HK, and ACPH) were monomorphic for each animal studied and no change in band migration pattern was observed. Banding patterns for the fourth system (EST) were observed to vary in the same individual. 3. The reversible induction of esterase variants in the tube feet of the same individual is reported. 4. The change was temperature dependent, and a period of 7-14 days acclimation was required to induce the pattern alteration.