The rainbow trout (Oncorhynchus mykiss) Mx1 promoter. Structural and functional characterization.
Mx1 gene expression was studied in the rainbow trout (Oncorhynchus mykiss) gonad (RTG) (fibroblast) cell line. RT-PCR analyses showed that both poly I:C and interferon containing supernatants induced expression of Mx1 in RTG cells. Kinetic analyses suggest that poly I:C acts indirectly through the production of interferons (IFN), as shown in other studies. By gene walking with trout genomic DNA the regulatory sequence of the Mx1 gene was cloned and sequenced. Sequence analysis showed that the 5' flanking region has a structure, which is typical for an interferon-induced gene promoter. Relative to the transcription start, it has a TATA box at -29 to -25, a 13 nucleotide interferon response element (ISRE) between -101 to -89, and a Sp1 binding site at -382 to -374. This region, with a single ISRE, is enough to induce strong expression of a luciferase reporter gene after stimulation of RTG cells with poly I:C. A time-course of induction of this reporter construct showed maximal expression (22-fold increase) after incubation with 100 microg mL(-1) poly I:C for 48 h. An optimized method of transient transfection of RTG cells is also described.
Department of Zoology, University of Aberdeen, UK. b.collet@abdn.ac.uk
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Secombes
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D019204:GTP-Binding Proteins; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D017686:Oncorhynchus mykiss / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D064547:Myxovirus Resistance Proteins; D011506:Proteins; D004247:DNA; D008156:Luciferases; D019204:GTP-Binding Proteins