Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with beta-naphthoflavone-treated fish

Induction of cytochrome P4501A (CYP1A) in fish is an important biomarker in marine monitoring programmes but a number of factors complicate interpreta tion of data based on catalytic activity. To provide additional analytical tools, we have cloned and sequenced entire (dab) and partial cDNAs (flounde r, turbot, sand eel) from several fish species. A detailed analysis compari ng the new sequences to those on the database (13 sequences) is presented a nd identifies an invariant, teleost-specific sequence (195-IVVSVANVICGMCFGR RYDH-214) which might be the basis for production of a species cross-reacti ve antibody. Northern and slot blots of fish RNA (sand eel, plaicc, turbot, flounder and dab) showed extensive cross-species hybridisation with each o f the cDNAs (sand eel, plaice, turbot, flounder and dab). The exception was turbot RNA, which only gave adequate hybridisation when the turbot probe w as used. Attempts to normalise the hybridisation data to GAPDH mRNA were no t satisfactory since there were significant species differences in expressi on of this gene and expression was suppressed (20-40%) by beta -naphthohavo ne treatment. The CYP1A probes indicated induction levels relative to untre ated dab of: plaice (five-fold); turbot (12-fold); flounder (12-fold); and dab (10-fold). The study demonstrates the relative ease with which species- specific molecular probes can be generated and used. (C) 2001 Elsevier Scie nce Inc. All rights reserved.