Isolation of 5′-untranslational region of trout Cyp1A1 gene

Abstract

The genomic DNA was prepared from trout liver which was treated with 3-methycholanthrene, and cloned into lambda EMBL3 at BamHI site. The genomic library was constructed via infections of these recombinant phages into E. coli K802, and screened by the most 5′-portion of trout CYP1A1 cDNA. After the screening of 10 9 clones of the amplified library, 12 positive clones were isolated, and subjected to further screenings. The results of southern blot hybridization of genomic DNA prepared from the positive clone showed the presence of a single gene of CYP1A1, and 3.5 Kb PstI fragment that hybridizes with the most 5′-region DNA of CYP1A1 cDNA. The restriction map of PstI fragment was determined by the restriction digestion with various enzymes. The nucleotide sequence of the upstream genomic DNA of CYP1A1 was determined by DNA sequencing of exonuclease III unidirectionally deleted PstI fragment DNA using [ 35S]dATP. This paper presented the upstream genomic DNA of CYP1A1 contained a part of coding region which was about 351 base pairs (from ATG to PstI site at 3563).