Structure and promoter activity of the LpS1 genes of Lytechinus pictus. Duplicated exons account for LpS1 proteins with eight calcium binding domains.

The LpS1 genes of the sea urchin Lytechinus pictus are activated early in development in aboral ectoderm cells. They therefore have ontogenic properties similar to their counterparts in Stronglyocentrotus purpuratus, the Spec genes. Both gene families encode proteins belonging to the calmodulin superfamily as evidenced by the presence of distinct EF-hand (helix-loop-helix) domains. The presence of eight EF-hand domains in LpS1 proteins suggests that the LpS1 genes arose from a duplication of an ancestral Spec-like gene. The LpS1 genes were further analyzed to increase our understanding of the mechanisms underlying their evolution and activation in aboral ectoderm cells. Genomic DNA blot analysis showed two LpS1 genes, LpS1 alpha and LpS1 beta, which did not appear to be closely linked. LpS1 genomic clones were isolated by screening an L. pictus genomic library with an LpS1 cDNA clone, and partial gene structures for both LpS1 alpha and LpS1 beta were constructed. These revealed internal duplication of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Duplication of exon 1 in both genes suggested four different LpS1 proteins could be derived from the LpS1 genes. Primer extension to map the transcriptional initiation sites of the LpS1 genes and sequencing analysis showed there was little in common among the 5'-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF. A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5'-flanking DNA and 17 bp of 5'-untranslated leader sequence of the LpS1 beta gene were sufficient for correct temporal and spatial expression of reporter chloramphenicol acetyltransferase and lacZ genes in sea urchin embryos. Deletions at the 5' end to either 511 or 368 bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase activity and disrupted the exclusive activation of the lacZ gene in aboral ectodermal cells. Based on a lineage analysis among the LpS1 and Spec gene families and other related genes, we propose a model in which LpS1 genes evolved from a series of duplications of an ancestral Spec-like gene.