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Cloning and functional characterization of the p65 subunit of NF-κB from olive flounder (Paralichthys olivaceus)

Kong, Hee Jeong, Moon, Ju-Hyun, Moon, Ji-Young, Kim, Jae-Min, Nam, Bo-Hye, Kim, Young-Ok, Kim, Woo-Jin, Lee, Sang-Jun
Fish & shellfish immunology 2011 v.30 no.1 pp. 406-411
transcription factor NF-kappa B, polypeptides, Paralichthys olivaceus, messenger RNA, complementary DNA, bacterial artificial chromosomes, flounder, stress response, conserved sequences, expressed sequence tags, rapid amplification of cDNA ends, gene expression, amino acids, tumor necrosis factor-alpha, cDNA libraries, immune response, molecular cloning, gene expression regulation, sequence homology
NF-κB is a master transcription factor found in almost all cell types that responds to diverse cellular stimuli by activating the expression of stress response genes, including immune-related genes. cDNA encoding the p65 subunit of olive flounder (Paralichthys olivaceus) NF-κB (Po-p65) was isolated through an EST analysis of an olive flounder cDNA library, a screen of BAC library, and rapid amplification of cDNA ends (RACE). The cDNA for Po-p65 encodes a polypeptide 626 amino acids in length containing a well-conserved Rel-homology domain (RHD). The primary sequence of Po-p65 showed strong homology with p65 from perch and zebrafish (82.7 and 64.4%, respectively), and shared 43.4–42.1% homology with p65 from other species, including mammals, while the N-terminal RHD of Po-p65 showed strong identity (95.6–67.8%) with that of other species. Po-p65 mRNA expression was detected in all flounder tissues examined. The over-expression of full-length Po-p65 (Po-p65f), but not of a Po-p65 C-terminus deletion mutant (Po-p65ΔC), stimulated κB element-driven reporter (κB-luc) activity in a dose-dependent manner and regulated the expression of p65 target genes, including TNF-α and IκB-α, in HINAE olive flounder cells. Po-p65f translocated to the nucleus following stimulation with poly I:C in HINAE cells. Together, these results suggest that Po-p65 is evolutionarily and functionally conserved in flounder and mammals and may provide clues to the detailed molecular mechanism(s) underlying immune response regulation in flounder.