Genomic organisation of the channel catfish Mx1 gene and characterisation of multiple channel catfish Mx gene promoters.
In order to further characterise channel catfish (Ictalurus punctatus) Mx1, studies were initiated to amplify and clone the Mx1 promoter into a reporter vector, pGL3basic. Initially the Mx1 gene was amplified from genomic DNA and was found to have 12 exons and 11 introns, spanning a region over 6 kilobases (kb) in length. The Mx1 promoter was amplified using genome walking and during this process four additional Mx promoters were identified, suggesting the presence of five Mx genes in the channel catfish. All five promoters possess an interferon stimulated response element (ISRE) and the Mx1 promoter possessed two potential NF-kappabeta transcription sites. Following cloning each construct was transiently transfected into COS-7 and EPC cells for 24h and treated with 5 microg/ml poly I:C for 24h. An increase in expression of the reporter gene in response to poly I:C was noted in both cell lines in the pGL3Mx1 construct only. However, the reporter gene was also constitutively expressed in these cells. Constitutive expression was also observed in channel catfish ovary cells transiently transfected with pGL3Mx1 only. Treatment with 5 microg/ml poly I:C did not increase this expression, which may be due to high levels of cell death in this difficult to transfect cell line. The constitutive expression observed implies that a repressor element is missing in the 390 base pair sequence of the Mx1 promoter used in this study. These results suggest that only channel catfish Mx1 is involved in the type I interferon pathway and that the presence of an ISRE in a regulatory region is not necessarily indicative of a role in the type I interferon response.
Plant
Karen P
KP
Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA. kplant@uidaho.edu
Thune
Ronald L
RL
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D029941:Fish Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D023061:Gene Order; D016678:Genome / Q000235:genetics*; D007059:Ictaluridae / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D011070:Poly I-C / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*
D029941:Fish Proteins; D064547:Myxovirus Resistance Proteins; D008156:Luciferases; D019204:GTP-Binding Proteins; D011070:Poly I-C