DURICA, D.S.*; KUPFER, D.; NAJAR, F.; SO, S.; TANG, Y.; GRIFFIN, K.; HOPKINS, P.M.; ROE, B.; University of Oklahoma; University of Oklahoma; University of Oklahoma; University of Oklahoma; University of Oklahoma; University of Oklahoma; University of Oklahoma; University of Oklahoma: Limb Regeneration and Expressed Sequence Tag Sequencing in Fiddler Crab

We have constructed several directional and randomly-primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab (Uca pugilator) limb regeneration. Data from these libraries is being assembled into an on-line database (http://www.genome.ou.edu/crab) that is both BLAST and keyword searchable; the data set will also be available through GenBank. The first characterized library was made from mRNA isolated fours days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of approximately 1500 cDNA clones led to assignment of 485 contigs and 429 singlets, for a total of 914 sequences. Of these, approximately 63% showed no homology on database searching. Analysis of these unclassified sequences relative to sequencing with 5� or 3� specific primers showed approximately equal numbers in each category, suggesting that the �no hit� clones may contain a large number of new genes. Biological assignments for the approximately 37% of ESTs based on KEGG (Kyoto Encyclopedia of Genes and Genomes) are broken down into metabolism or regulatory categories, and a detailed list of assignments based on the NCBI COG (Clusters of Othologous Groups) outline format is available. These ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes. Homologs to arthropod proteins involved in retinoid/terpenoid metabolism have also been identified. To investigate potential ecdysteroid-responsive candidate genes, hormone induction experiments are currently in progress, using primer sets designed from database sequence information and quantitative real-time PCR.