Brian T. Livingston

Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development.

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated “tyrosine switch.” It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis.

Tracing misalignment and unequal crossing over during evolution of the repetitive region of the SM50 gene in sea urchins.

The location of misalignment and unequal crossover involved in concerted evolution of tandemly repetitive sequences is difficult to document owing to the homogeneity of sequences that are subject to this process. However, the repetitive domain of the SM50 gene in sea urchins contains variation, within the gene itself, between alleles, and between species that has allowed us to determine where misalignment and unequal crossing over occurred during evolution of this gene. We have therefore analyzed the SM50 repeat regions in a variety of species to determine where recent changes in repeat numbers have occurred, and from this have deduced the mechanisms that lead to these changes. We next tried to determine whether recent misalignment and unequal crossover has produced allelic variation in current populations of sea urchins. We found SM50 alleles within three species that have different numbers of repeats. This marks the first reported documentation of allelic variation in the number of repeats in the SM50 gene. We also show how a single unequal crossover event could have produced the allelic variation. We have found that substitutions and small deletions in the sequences within the repeats can substantially affect how misalignment occurs, resulting in different patterns of repeats after concerted evolution.

Identification of cis-regulatory elements involved in transcriptional regulation of the sea urchin SpFoxB gene

The SpFoxB gene is transiently expressed first in the mesoderm, then in the endoderm and oral ectoderm during sea urchin gastrulation. Perturbations of a number of proteins involved in endomesoderm specification have been shown to alter the mRNA levels of SpFoxB, but the cis‐regulatory elements required for expression of SpFoxB have not been examined. In order to investigate this, we have screened the SpFoxB gene for sequences that can drive its expression. Both positive and negative cis‐regulatory elements were found to be present. An enhancer was found that contains four GATA sites and four YY1 sites clustered within 210 base pairs (bp), as well as three lef/tcf binding sites. Electrophoretic mobility shifts indicate that the lef/tcf sites bind a complex of proteins that include β‐catenin in early cleavage, but not during subsequent stages of development. The GATA and YY1 sites bind nuclear proteins prior to SpFoxB transcription, and this binding diminishes coincident with cessation of transcription. Deletion of the GATA/YY1 sites causes a significant decrease in transcription. The DNA binding site of the SpFoxB protein has been determined, and Fox binding sites are found within the 5′ UTR of SpFoxB.