Cloning, sequencing, and tissue expression of CYP3A27, a new member of theCYP3A subfamily from embryonic and adult rainbow trout livers

Screening of lambda gt11 and lambda gt22A cDNA libraries of livers from adu lt females and embryos of rainbow trout (Oncorhynchus mykiss), respectively , using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodie s showed that there were identical cDNAs of 1802-bp nucleotides with open r eading frames coding for proteins containing 518 amino acids (59,206 Da, pI = 6.39), The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P4 50 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence o f rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with mo nkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24, Marked differences in sex, age, and tissue expression of CY P3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in th e fish examined. Southern blot analysis of restriction enzyme-digested rain bow trout genomic DNA demonstrated that multiple CYP3A27-related genes exis t in rainbow trout. (C) 1998 Academic Press.