SHAFER, T.H.; University of North Carolina, Wilmington: Expressed sequence tag sequencing to identify genes involved in exoskeleton calcification in the blue crab

A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including the discovery of genes for cuticle proteins regulating calcification. One of the expression libraries sequenced was from hypodermal tissue, the epithelium that deposits the cuticle. The RNA used for cDNA synthesis was pooled from arthrodial and dorsal hypodermis of both pre-ecdysis (D2) and 3-h postecdysis crabs. This ensured representation from both calcifying and non-calcifying cuticle regions and from cuticle layers deposited both before and after exuviation. The EST data have been mined for cuticle protein sequences. First, we searched for known cuticle-specific motifs, the Rebers-Riddiford chitin-binding sequence and a mineralized-tissue motif described by Andersen. Second, we checked the BLAST annotations and the GO analysis of the entire EST project for evidence of similarity to known (often insect) cuticle proteins. Finally, all crustacean cuticle protein sequences in NCBI were placed into an alignment-based dendrogram resulting in broad similarity groups. Then BLAST was used to search the EST data for significant homologies to each group. In all, the database appears to contain approximately 70 contigs or singlets representing transcripts of cuticle proteins. Forty-five distribute among ten clusters of very similar transcripts, possibly representing alternate splicing or recent gene duplications, while the rest share less similarity. We have obtained complete sequences for about 25 of the transcripts, and we are determining gene expression patterns across the molt cycle in calcifying versus non-calcifying cuticle regions. The combination of homology analysis and gene expression analysis allows us to infer possible functions in cuticle synthesis and calcification.