Brian T. Livingston

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites.

The mechanism of determination of early embryonic cells has been investigated using sea urchin embryos. An efficacious method of isolating blastomere pairs from the animal or vegetal half of sea urchin embryos was developed. The overt differentiation of separated animal and vegetal blastomere pairs resembles that of separated animal and vegetal hemispheres isolated by manual dissection. Treatment of animal blastomeres with LiCl caused them to display a morphology resembling that of isolated vegetal blastomeres. The effects of separation of animal and vegetal blastomeres and of treatment of animal blastomeres with LiCl were examined at the molecular level using gut alkaline phosphatase and a spicule matrix protein RNA as markers of differentiation. Histochemical staining and in situ hybridization studies showed that these markers are normally only expressed in vegetal blastomeres but that their expression can be evoked in animal blastomeres by treatment with LiCl.