SEQUENCE AND EXPRESSION OF A REBERS-RIDDIFORD 3 CONTAINING PROTEIN IN THE BLUE CRAB, CALLINECTES SAPIDUS

The blue crab, Callinectes sapidus, is an excellent model organism for studying biomineralization, the process by which living organisms form inorganic solids, due to its regular molting and precise temporal and spatial controls of cuticle calcification. A transcript believed to be involved in cuticle formation was selected from an Expressed Sequence Tag (EST) library. Once the full-length sequence was obtained and the transcript was virtually translated into a protein, the transcript was analyzed both for the presence of Rebers-Riddiford motifs and its expression pattern. Proteins containing Rebers-Riddiford consensus sequences have been shown to bind chitin in vitro. Alignment with a lobster protein, as well as two known insect cuticle proteins, revealed the presence of a Rebers-Riddiford 3 consensus sequence in the transcript, making it the first C. sapidus cuticle protein identified as having an RR-3 motif. Transcript expression analysis was conducted utilizing reverse transcriptase PCR, real-time PCR, northern blotting, and in situ hybridization. While the expression data is not conclusive, the transcript appears to be the only C. sapidus protein expressed both pre- and post-molt, in mid-dorsal (calcifying) as well as arthrodial (non-calcifying) tissue, as well as the first RR-3 containing transcript shown to be expressed pre-molt.