A reverse transcription-polymerase chain reaction (RT-PCR) approach for cloning Ah receptors from diverse vertebrate species: Partial sequence of an Ah receptor from the teleost Fundulus heteroclitus

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons. AhR proteins are expressed in most vertebrate animals, including teleost and elasmobranch fish, but the relationship between piscine and mammalian Ah receptors is uncertain. In the present studies, cloning of an Ah receptor cDNA from the teleost Fundulus heteroclitus was pursued using a reverse transcription-polymerase chain reaction (RT-PCR) approach. Degenerate oligodeoxynucleotides were designed based on conserved regions of mammalian Ah receptors and the related PAS proteins Per, ARNT and Sim. The oligonucleotides were used as primers in the RT-PCR to amplify a portion of an AhR expressed in Fundulus liver. A fragment of approximately 700 bp was amplified and directly sequenced. The deduced amino acid sequence of this fragment (212 residues) shares significant sequence identity with the PAS domains of the mouse (64%), rat (64%) and human (62%) Ah receptors. These data reveal conservation of Ah receptor structure between mammals and fish, vertebrate groups separated by over 400 million years of evolution. The successful use of RT-PCR to amplify a Fundulus AhR establishes the utility of this approach in the phylogenetic analysis of Ah receptor structure and function