The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system.
Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in lambda FIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100 nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.
Williams
T D
TD
School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK.
Lee
J S
JS
Sheader
D L
DL
Chipman
J K
JK
D000818:Animals; D001483:Base Sequence; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D005091:Exons; D005432:Flounder / Q000235:genetics*; D005809:Genes, Regulator; D017930:Genes, Reporter; D005821:Genetic Techniques; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data
D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1