Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization |
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Authors: | Yuuki Inoue Tsubasa Saito Mariko Endo Chiaki Haruta Takeshi Nakai Tadaaki Moritomo Teruyuki Nakanishi |
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Affiliation: | (1) Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-8510, Japan;(2) Department of Biosystems Science, School of Advanced Sciences, The Graduate University for Advanced Studies (Sokendai), Hayama 240-0193, Japan;(3) Aburatsubo Marine Park, Misaki, Miura, Kanagawa 238-0225, Japan |
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Abstract: | Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP31 and MIP32 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP31 and MIP32 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP31 and MIP32 contain four exons and three introns, and MIP31 and MIP32 shared the same intron/exon organization with that of human. The MIP31 and MIP32 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP32 gene was much lower than the Trsc-SCYA107 and MIP31 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP31 and MIP32 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP32 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP31 expression was observed at 3–12 h poststimulation only with PMA. |
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Keywords: | Banded dogfish CC chemokine Suppression subtractive hybridization RT-PCR cDNA |
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