Functional characterisation of the Japanese flounder, Paralichthys olivaceus, Mx promoter.

The Japanese flounder, Paralichthys olivaceus, genome appears to encode a single Mx gene based on Southern blotting and previous cDNA studies. The 5' flanking region of the Japanese flounder Mx gene was cloned and analysed for its regulatory regions. A TATA box (-24 to -30), two interferon-stimulated response elements (ISREs) (-69 to -80 and -508 to -521) and two Sp1 sites (-563 to -572 and -994 to -1003) were identified relative to the transcription start site. The effects of various stimuli, as well as the effects of various promoter mutations, were investigated in a transient expression system using Japanese flounder (hirame) natural embryo (HINAE) cells and luciferase reporter gene constructs. Although not sensitive to LPS, ConA or PMA, reporter gene expression increased more than 10-fold after stimulation by polyinosinic:polycytidilic acid (poly I:C), an established inducer of interferon. Deletion mutational analyses revealed the ISRE closest to the transcription start site to be crucial for promoter activity. The distal ISRE, despite its relatively distant location, contributed to induce maximal promoter activity, but when alone was not sufficient by itself to elicit any significant promoter activity. An electrophoretic mobility shift assay confirmed the binding of transcription factors to both ISREs. Induction of luciferase by poly I:C was inhibited by 2-Aminopurine, a protein kinase (PKR) inhibitor, in a dose-dependent (1-10 mM) manner, suggesting that PKR may be required as a signal transducer for type I IFN signaling in fish. This Mx reporter assay may be useful for quantifying the responses and elucidating the regulation pathways of IFN type I.