Expressed sequence tag analyses of the Japanese flounder, Paralichthys olivaceus

Abstract

We have constructed nine cDNA libraries from the Japanese flounder Paralichthys olivaceus for expressed sequence tag (EST) analysis; these include liver, spleen, and skin of healthy fish, three stimulated leukocyte cDNA libraries, two stimulated kidney libraries, and an Ig+ leukocyte library. From the cDNA libraries of liver, spleen, and skin, taken from healthy Japanese flounder, a total of 670 clones (511 independent clones) were obtained. Of the 511 clones, 236 (46%) were identified as known genes using the BLAST database. The leukocyte libraries were obtained from a cloned population of Japanese flounder, the first of which was constructed from leukocytes infected with Hirame rhabdovirus (HRV). From this library 1237 cDNAs (896 independent clones) were obtained, from which 350 (39%) were identified as known genes. cDNAs that were upregulated in HRV-infected Ig+ leukocyte cells of the Japanese flounder were identified by differential hybridization using subtracted and un-subtracted cDNA probes. This procedure yielded 160 cDNA clones (50 independent); 24 of the 50 clones were identified as known reported genes. The other two leukocyte cDNA libraries were obtained from Japanese flounder leukocytes induced with either a combination of concanavalin A (ConA) and phorbol myristate acetate (PMA) or lipopolysaccharide (LPS). The former yielded 550 cDNAs, 47% of which were identified as known genes; the latter yielded 94 cDNAs, 54% of which were identified as known genes. A cDNA library from Japanese flounder kidney treated with ConA and PMA produced 381 clones, 68% of which were identified as known genes, whereas a cDNA library from kidney of Japanese flounder treated with LPS yielded 373 clones, 76% of which were homologous to known genes. A total of 2855 clones from the nine cDNA libraries were sequenced. Of these clones, 1464 (51%) have some similarity or identity to known sequences in the DNA/protein databases. The identified genes were classified into seven categories according to gene function: cell division, cell signaling/communication, cell structure/mobility, cell/organism defense, gene/protein expression, metabolism, and unclassified. The ratios of these seven categories were different within these libraries, as was the ratio of functional genes between ConA/PMA, LPS-treated leukocytes, and virus-infected Japanese flounder leukocytes. These results indicate that using different stimulation techniques on a given cell type is a good methodology for the isolation of different sequences by EST analysis. Many interesting biodefense and immunoresponse-related genes were revealed by the EST analysis conducted on each library. Together, these results further demonstrate that EST analysis is a powerful and useful technique for the identification and characterization of fish genes.