Journal
MARINE ENVIRONMENTAL RESEARCH
Volume 50, Issue 1-5, Pages 1-6Publisher
ELSEVIER SCI LTD
DOI: 10.1016/S0141-1136(00)00053-2
Keywords
flounder; cytochrome P450; CYP1A; reporter gene assay; promoter
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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 100nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility. (C) 2000 Elsevier Science Ltd. All rights reserved.
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