Meeting Abstract

P2.37  Monday, Jan. 5  A new family of crustacean cuticle proteins possibly related to mineralization of pre-exuvial cuticle SHAFER, T.H.*; KNAPP, W.E.; GOLUS, J.M.; University of North Carolina, Wilmington shafert@uncw.edu

A large group of closely related cDNAs was discovered by mining a database of ESTs derived from the hypodermis of the blue crab, Callinectes sapidus. Complete sequencing showed that these cDNAs code for proteins containing the chitin-binding Rebers-Riddiford-1 motif and a conserved sequence of 14 amino acid residues just C-terminal to the RR-1. This sequence and arrangement are not known from any other crustacean or insect cuticle protein except C. sapidus CP14.1 (reported previously and now recognized as a member of this new protein family) and calcification-associated soluble matrix protein-2 from the crayfish Procambarus clarkii. Transcription of these genes as determined by real-time qPCR is high only in pre-molt hypodermis synthesizing exocuticle that will mineralize post-molt rather than arthrodial membrane that remains soft. Interestingly, reverse transcriptase-PCR revealed two sizes of mRNAs from these genes, suggesting the presence of an unspliced intron. Sequencing the PCR products as well as genomic DNA using the same primers confirmed this. The intron is not efficiently spliced from some of the RNAs when transcribed post-molt, a period when the total transcription is very low. In other cases, transcripts containing unspliced introns were also present in hypodermis making arthrodial membrane, again where total transcription is very low. All unspliced transcripts would translate into truncated peptides because of in-frame stop codons in the introns. Though the function of this new family of proteins is not known, its expression pattern suggests a role in pre-exuvial exocuticle mineralization. A double-strand RNA reagent has been synthesized to knock out these transcripts, and RNAi loss-of-function assays are being attempted.