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In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Lysozyme is a widely distributed enzyme located in the serum, skin mucus, and other organs of fish, which is responsible for catalyzing the hydrolysis of the cell walls of most bacteria. A c-type of lysozyme cDNA was cloned from a kidney cDNA library of the Japanese flounder (Paralichthys olivaceus). The cDNAs consisted of 612 bp, which coded for 143 amino acid residues. The deduced amino acid sequence of Japanese flounder c-type lysozyme possessed 72.9%, 57.4%, and 65.4% identities with rainbow trout, chicken, and human c-type lysozymes, respectively. Comparison of the c-type lysozymes showed that the catalytic residues, the residues binding to sugar chains, and cysteine residues were completely conserved. Northern blot analysis indicated that the c-type lysozyme gene is apparently transcribed in the head kidney, posterior kidney, spleen, brain, and ovary of healthy flounder. When flounder were experimentally infected with Edwardsiella tarda, quantities of the c-type lysozyme mRNA increased in the head kidney, spleen, and ovary of the flounder.