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912 Gene discovery in Carcinus maenas and Homarus americanus via expressed sequence tags.pdf

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912 Gene discovery in Carcinus maenas and Homarus americanus via expressed sequence tags

Gene discovery in Carcinus maenas and Homarus americanus via expressed sequence tags David W. Towle1 and Christine M. Smith Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USA Synopsis Expressed sequence tags (ESTs) were produced for normalized cDNA libraries prepared from several tissues of 2 marine crustaceans, the green shore crab Carcinus maenas and the American lobster Homarus americanus. Tissues repre- sented in the Carcinus library were anterior and posterior gills, hypodermis, heart, hepatopancreas, antennal gland, brain, testis, and skeletal muscle, obtained from animals acclimated to 35 and 10‰ salinity. Tissues represented in the Homarus library were gill, epipodite, branchiostegite, heart, ovary, testis, antennal gland, skeletal muscle, hepatopancreas, and brain, obtained from intermolt and post-molt animals. Directional libraries from oligo-dT-primed cDNA were constructed in the pCMVsport6.1 vector and normalized by self-subtraction at 2 different Cot values. Randomly picked clones were single-pass sequenced from the 50 end. Raw sequence data were trimmed and prepared for submission to dbEST using phred, cross-match, and blastx protocols embedded within trace2dbest software. A total of 4604 Homarus and 12 401 Carcinus ESTs produced 540 and 2651 clusters, respectively, as determined by TIGR Gene Index Clustering software. Gene Ontology analysis with reference to a Drosophila melanogaster database using GOblet revealed 187 positive hits (35%) with the Homarus clusters and 1037 positive hits (39%) with the Carcinus clusters. Combining the number of assembled sequences with the number of singlets obtained after cluster analysis suggested that transcripts representing as much as 25% of the total number of genes in Carcinus have been isolated. Introduction The green shore crab Carcinus maenas is a highly inva- sive portunid crab, originating in coastal regions of northern Europe and now occupying m