Functional analysis of promoter region from eel cytochrome P450 1A1 gene in transgenic medaka

Transcription of the CYP1A1 gents in mammals and fish is stimulated by poly aromatic hydrocarbons. DNA sequencing analysis revealed that CYP1A1 gene in eel (Anguilla japonica) contains two kinds of putative cis-acting regulato ry elements, XRE (xenobiotic-responsive element) and ERE (estrogen-responsi ve element). XRE. is known as the enhancer that is responsible for the indu cibility of the gents of CYP1A1 and some other drug-metabolizing enzymes. I n the eel CYP1A1 gene, XRE motifs are distributed as follows: five times in the region from -2136 to -1125 bp, XRE(-6) to (-2);; once in the proximal basal promoter region, XRE(-1); and once in the first intron, XRE(+1). The region between XRE(-2) and XRE(-1) contains three EKE motifs. To investigat e the function of the cis-acting regulatory elements in the eel CYP1A1 gene , recombinant plasmids prepared with its 5' upstream sequence and the struc tural gene for luciferase were microinjected into fertilized eggs of medaka at the one-cell stage. Hatched fry were treated with 3-methylcholanthrene, and the transcription efficiency was assayed using competitive polymerase chain reaction analysis. Deletion of the region containing the five XREs, X RE(-6) to XRE(-2), and the point mutation of XRE(-1) reduced the inducible expressions by 75% and 56%, respectively, showing apparent dependency of th e drug induction on the XREs. Constitutive expression, however, was not sig nificantly affected by deletion or disruption of the XREs. When the region between XRE(-2) and XRE(-1) containing no XREs but three ERE motifs was int ernally deleted, the inducible expression and the constitutive expression w ere reduced by 88% and 75%, respectively. Replacement of this region with a partial fragment of eel CYP1A1 complementary DNA, with slight alteration o f the distance between the five XREs and XRE(-1), reduced the inducible exp ression and the constitutive expression by 91% and 60%, respectively. These results strongly suggest that not only XRE but also other regulatory eleme nts, possibly ERE, play an important role in induced and constitutive expre ssions of the eel CYP1A1 gene.