EXPRESSED SEQUENCE TAGS (ESTS) ANALYSIS OF THE LEUKOCYTES OF JAPANESE FLOUNDER, Paralichthys olivaceus, TREATED WITH CON A/PMA

Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan

We constructed a cDNA library of Japanese flounder leukocytes treated in vitro with T cell mitogen of concanavalin A (Con A) and tumor-promotor of phorbol 12-myristate 13-acetate (PMA) in order to get cytokines and biodefense-related proteins. The 300 cDNA clones were partially sequenced from the 5' and/or 3' ends of inserts from the Japanese flounder leukocyte cDNA library. About 139 (46.3%) cDNA clones represented known genes in the public databases, whereas the remaining 164 (54.7%) of the clones did not show any significant homology with the sequences in the public database. The average size of the sequenced length was 882bp. 0ver 60% of the known genes were identified as putative biodefense genes or genes associated with immune response including tumor necrosis factor alpha, Fas-antigen ligand, interleukin 8, transcription factor for immune related genes, stress protein, activators of immune cells, and membrane surface protein of immune cell. We have carried out subtractive cloning to isolate differentially expressed sequences in the stimulated leukocyte cells of Japanese flounder. The 72 of positive clones were obtained from the cDNA library after hybridization with subtractive fragments as probe. The result of sequencing and homology search of the positive clones yielded 37 (51.4%) identified known genes, and 35 (48.6%) unknown genes. 20 clones had homologies with immune associated genes.