Natural spawning of a Hawaiian sea urchin,Tripneustes gratilla

Observations of natural spawning events are rare but critically important for the field of fertilization ecology. For broadcast spawners, knowing the natural behavior of spawning, including proximity of animals and the timing of spawning, is essential for informing spawning experiments and directing future study. Here we describe a natural spawning event that took place on the island of Maui in the winter of 2010. Groups of Tripnuestes gratilla were observed to spawn in the late afternoon shortly after a local high tide that was a lower high of a mixed, semidiurnal tide cycle. Urchins appeared to increase the probability of fertilization by ascending to the local high points of the reefs and spawning within groups of 2–5. Although the majority of the urchins that spawned were T. gratilla, two individuals of Echinothrix diadema were observed to spawn at the same time, raising questions about potential hybridization in the wild.     

Length and sequence heterogeneity of the histone gene repeat unit of the sea urchin, S. purpuratus

Histone gene repeats in S. purpuratus are shown to be of variable length and sequence. Two recombinant plasmids containing the full-length 6.3 kb histone repeat unit are found to differer in length at two sites in the repeating structure and in the occurrence of two restriction enzyme recognition sites. Variation in repeat length is also demonstrated in the unfractionated DNA of five sea urchins and in a sample of DNA enriched for histone gene sequences by density gradient methods. The repeats in each individual are of a very limited number of major classes, which may differ from one another in overall length or in distribution and presence of particular restriction enzyme sites. Variations are found to occur at many regions of the repeat; some have been mapped specifically to spacer regions. Repeats may differ dramatically from individual to individual since there is no one type of repeat class common to all, although the absolute length differences of the repeats that are found are small.     

Nucleotide sequence of a 5S ribosomal RNA gene in the sea urchin Lytechinus variegatus.

The nucleotide sequence of a cloned DNA fragment corresponding to a gene for 5S ribosomal RNA from the sea urchin Lytechinus variegatus has been determined. This sequence is representative of the dominant species of 5S rRNA labelled in vivo with 32pO4 during the cleavage stage of Lytechninus embryonic development.     

Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer

Summary A common polymorphism of the early embryonic histone-gene repeat ofStrongylocentrotus purpuratus is a 195-bp insertion within the H4-H2B spacer. The sequence, found as an insert in histone-gene repeats of 6 of 22 individuals screened, is also found at approximately 50 sites elsewhere in the genome of every individual. We compare the sequences of the histone-gene spacers that do and do not contain the insert. The insert is found not to have transposon-like features, and no sequence in the original spacer has been duplicated to flank the insert. There is, however, a hexanucleotide sequence that is repeated three times at one end of the insert, and the element has inserted between direct repeats of 5 bp that were present in the original spacer. One of the copies found outside the histone gene cluster was cloned and sequenced and is compared with the insert. Again, no transposon-like features are evident. Regions flanking the homologous sequence in this clone were used as hybridization probes in whole-genome blots. Results indicate that the 195-bp sequence insert is itself embedded within a larger element that is repeated within the genome. Therefore, only a portion of a larger repetitive sequence has integrated into the histone-gene spacer. The sequence features of the insert, although not typical of mobile elements, may be representative of other illegitimate recombination events.     

Molecular characterization of TgHBox4, a Drosophila Abd-B homolog found in the sea urchin Tripneustes gratilla

We have isolated and sequenced a cDNA clone that, as judged by the sequence of the homeobox region, encodes a sea urchin homolog of the homeobox containing the gene Abdominal-B of Drosophila. The total length of the cDNA is 3634 nucleotides and includes an open reading frame, which encodes a protein that is 32,321 Da. The N-terminal region of the homeodomain includes consensus sequences found in some of TgHBox4's Abdominal-B relatives. A genomic clone representing the 5' part of the message was also isolated. This clone and a previously isolated clone were found to represent the full-length cDNA sequence. We have also raised antibodies against a bacterially expressed portion of the TgHBox4 protein and used them to determine the location of TgHBox4 proteins during development. The protein displays ubiquitous expression early in development but becomes more restricted, to posterior regions, late in embryogenesis. Thus, in contrast to its Abd-B homologs in bilateral metazoans, TgHBox4 is probably not involved in pattern formation but may have a posterior-defining role late in embryogenesis.     

Studies on the regulation of ribosomal RNA synthesis in sea urchin development.

Cells of sea urchin hatching blastulae and gastrulae when reaggregated together do not influence each other with respect to the rate of rRNA synthesis. Extracts from unfertilized eggs and embryos inhibited rRNA synthesis by gastrulae. However, the inhibition was equally strong with extracts from stages that have a low rate of rRNA synthesis (eggs, cleavage embryos) as with extracts from stages that have a high rate of rRNA synthesis (oocytes, gastrulae). Synthesis of ppGpp is not detected at any of the investigated developmental stages.     

Evolving sea urchin histone genes-nucleotide polymorphisms in the H4 gene and spacers ofStrongylocentrotus purpuratus

Summary We present a comparison of spacer and coding sequences of histone gene repeats from fourStronglycocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions: tranversions: single-base-pair insertions/deletions was 322. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)_n and (CT)_n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of everyS. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pariwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1–2 million years necessary for accumulation of the variation. The level of sequence variation found within theS. purpuratus population, for both tandemly repeated and single-copy genes, is 25%–50% of that found betweenS. purpuratus andS. drobachiensis.     

DNA sequence analyses of the ribosomal spacer regions in theTriticeae

Two regions of the ribosomal DNA (rDNA) were sequenced from a range of species from the tribeTriticeae. One region, the central spacer, was found to be more divergent in sequence than the other, the 18 S-spacer junction. Both regions contained sequences 20–30 bp long which were more highly conserved than the remainder of the region and their possible significance in rDNA expression is discussed. Phenetic relationships based on the sequence data were generally consistent with the relationships based on other criteria. Species possessing the S, E, J₁J₂, D, and B genomes clustered together, with the H genome species being the most distinct of those examined. The R, P, and V genome species occupy an intermediate position in the overall pattern of relationships. Some relationships differed in detail from those established by other parameters, for example the position of the N genome species, and explanations for discrepancies of this type are discussed.     

The role of the sea urchin,Tripneustes gratilla (Linnaeus), in decomposition and nutrient cycling in a tropical seagrass bed

The sea urchin,Tripneustes gratilla, which feeds mainly on living leaves of the seagrass,Thalassia hemprichii, was studied in its habitat on the southern coast of Papua New Guinea, and its roles in decomposition and nutrient cycling in a seagrass bed were assessed through the excretion of ammonium and metabolism of feces produced by the sea urchin. Carbon content of the fresh feces (21% of dry weight) was similar to that of intact dead leaves of the same species (22–23%). Carbon/nitrogen and carbon/phosphorus ratios of the feces (21.7 and 466, respectively), however, were significantly lower than those of the dead leaves (25.9–27.7 and 656–804, respectively), indicating that the feces retain more nitrogen and phosphorus in comparison with carbon. Net consumption of ammonium and orthophosphate typically concurred with oxygen consumption during dark incubation of both the dead leaves and the sea urchin feces. Compared with the same oxygen consumption rate, however, the dead leaves consumed more orthophosphate than the feces. Under sunlight, dead leaves showed a net accumulation of carbon by epiphytic algae, while the feces showed a carbon loss. Ammonium excretion by this sea urchin (1.7–5.4 mg nitrogen/individual/day) would thus appear to make a significant contribution to nitrogen recycling since biological communities associated with dead leaves and sea urchin feces tend to demand an external supply of nitrogen, such as ammonium.     

Length heterogeneity in a region of the human ribosomal gene spacer is not accompanied by extensive population polymorphism

We carried out a restriction enzyme analysis of human ribosomal DNA structure on total placental DNA using the Southern (1975) method. Studies on a single individual using HindIII, PstI, HpaI and BglII revealed that a region of the non-transcribed spacer near the 3′ end of the 28 S gene was heterogeneous in size. Four fragment classes were detected in this individual. Adjacent classes differed in size from one another by about 0·8 × 10³ bases. Analysis of 19 additional placental samples and four cell lines revealed no fragment classes other than those detected in the original sample. Mixing experiments carried out with all 20 placental DNA samples provided further evidence for the discrete nature of the population polymorphism in the length of this region of the spacer. This finding contrasts sharply with the almost continuous nature of the population polymorphism in the length of a region of the non-transcribed spacer in Xenopus laevis.     

Structure and expression of the polyubiquitin gene in sea urchin embryos.

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3' noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock-inducible during embryogenesis.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.