COBLENTZ, F. E.*; TOWLE, D. W.; SHAFER, T. H.; University of North Carolina at Wilmington; Mount Desert Island Biological Laboratory; University of North Carolina at Wilmington: Expressed sequence tags from normalized cDNA libraries prepared from gill and hypodermal tissues of the blue crab, Callinectes sapidus

Two cDNA libraries were constructed from gill and hypodermal tissue of the blue crab, Callinectes sapidus to produce expressed sequence tags (ESTs). Gill RNA was isolated from anterior and posterior gills dissected from adult intermolt crabs acclimated to salinities of 35 or 5 parts per thousand and pooled. Hypodermal RNA was isolated from arthrodial (uncalcified) and mid-dorsal (calcified) hypodermis dissected from adult pre and post molt crabs and pooled. The quality of the pooled RNA from gill and hypodermis was assessed on a RNA gel and each pooled RNA sample was used to construct and normalize a cDNA library. Randomly chosen cloned cDNAs were sequenced from their 5′-ends, and vector sequences as well as low quality sequences were removed. The resulting 11811 EST sequences had an average length of 652 bases. They were assembled into contigs using Paracel Transcript Assembler software. This produced 883 contigs (304 of them were exclusively gill, 216 exclusively hypodermis) and 1293 singlets which together represent 2176 potential transcripts. Contigs and singlets were compared to the non-redundant protein database at NCBI using BLASTx and annotated with descriptions of the three most similar proteins. A web page is currently under development which will include the sequences in a format that is �BLASTable� and the annotations in a searchable format. In addition, the ESTs have been submitted to GenBank using trace2dbEST software (Parkinson, Anthony and Blaxter, unpublished software). The database of ESTs from this project has already been a valuable resource in gene discovery related to biomineralization. Funded by UNC Office of the President, Genomic Research Support Grant.