Molecular Cloning and Novel Repeated Sequences of a C-type Lysozyme Gene in Japanese Flounder (Paralichthys olivaceus)

Abstract

A clone of a DNA fragment of approximately 10 kb that included a c-type lysozyme gene of Japanese flounder (Paralichthys olivaceus) was isolated from a Japanese flounder genomic DNA library. This clone was subcloned, and the nucleotide sequence of the c-type lysozyme gene was determined. The flounder c-type lysozyme gene, which consisted of 3,617 bp, was found to be composed of four exons and three introns. The transcription start site was determined to be 44 bp upstream of the ATG codon by primer extension analysis. The number of exons and introns and the positions of the catalytic residues of the flounder gene were the same as those of the human and chicken c-type lysozyme genes. However, introns 1 and 3 of the flounder gene were shorter, and intron 2 was longer, than those in the other reported vertebrates genes. There were no Alu repeated sequences in the flounder lysozyme gene, as determined for the human lysozyme gene, but 45 repeated sequences were found in the second intron of the flounder lysozyme gene. We suggest that the length of intron 2 in the Japanese flounder c-type lysozyme gene depends on the presence of this repeated sequence. Furthermore, Southern blot analysis revealed that the c-type lysozyme gene was probably a single copy in the genomic DNA of homo-cloned Japanese flounder.

Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein

“…c-type lysozymes have been isolated from many vertebrates and insects (Prager & Jollès 1996, Qasba & Kumar 1997. In fishes, complete coding sequences of the c-type lysozymes of Japanese flounder (Hikima et al 1997) (GenBank #AB050469), turbot (GenBank #AJ250732), rainbow trout (Dautigny et al 1991) (GenBank #X59491), zebra fish (GenBank #AF402599) and common carp (GenBank #AB027305) have been reported. G-type lysozymes have also been characterized in geese, chickens, black swans and ostriches (Canfield & McMurry 1967, Simpson et al 1980, Schoentgen et al 1982, Nakano & Graf 1991, and gtype lysozyme full-length cDNA sequences have been reported from chickens (Nakano & Graf 1991), flounder (Hikima et al 2001), carp (GenBank #AB084624), mice (GenBank #AK009014) and humans (GenBank #BC029126).…”

Analysis on the Molecular Biologic Characteristics and Expression of Lysozyme C Separated from Oplegnathus fasciatus

“…In particular, it was reported that the number of the genes significantly increases after experiment testing bacterial attacks. For Paralichthys olivaceus, the gene was identified most abundantly in head kidney, spleen, and ovary [1].…”

“…In particular, it was reported that the number of the genes significantly increases after experiment testing bacterial attacks. For Paralichthys olivaceus, the gene was identified most abundantly in head kidney, spleen, and ovary [1].Oplegnathus fasciatus belongs to class of Chordata, Actinopterygii, and perciformes, and is temperate zone fish usually inhabiting in rock zones of coasts. They are distributed in all coastal areas of Korea, the Japanese coastal areas, and the Chinese coastal areas.…”

“…This suggests that the c-type lysozymes still basically function as a defensive mechanism. In Paralichthys olivaceus, the c-type lysozymes were predominantly identified in head kidney, posterior kidney, spleen, brain, and ovary [1],whereas the g-type lysozymes in head kidney, posterior kidney, spleen, skin, muscles, heart, and brain [8]. In Ctenopharyngodon idella, the cDNA of the c-type lysozymes were broadly expressed in all parts, of which they are the most highly expressed in the head kidney [13].…”

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, Anguilla japonica

“…An important innate immune component is the mucus layer on the surface of fish which serves as the first line of defense against invasion between fish and its aquatic environment [37]. It is well established that skin secretions of fish contain a number of innate humoral defence factors, including complement factors [47], lectins [3], proteases [20] and lysozymes [21,38]. It is revealed that the surface mucosa of some fish also contains a range of AMPs, such as those with the molecular weight (MW)\10 kDa from Misgurnus anguillicaudatus [10,32], Pardachirus marmoratus and Pardachirus pavoninus [24,42], Pleuronectes americanus [8], Salmo salar [29], Oncorhynchus mykiss [16,17], Morone chrysops and Morone saxatilis [28], Hippoglossus hippoglossus L. [5], Parasilurus asotus [33] and Myxine glutinosa [41].…”