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Chang Ching-yi; Smith, David R.; Prasad, V S; Sidman, Charles L.; Nebert, Daniel W.; Puga, Alvaro
Pharmacogenetics: December 1993
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We have analysed by heteroduplex formation (HF), single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and nucleotide sequencing the cDNAs of the Ahrb-l and Ahrd allelic forms of the aromatic hydrocarbon receptor (AhR) present in inbred strains of mice. The Ahrb-l allele, found in the C57BL and C57BR strains, encodes a 95 kDa receptor with an affinity for ligand 15—20 times higher than the affinity of the 104 kDa receptor encoded by the Ahrd allele, found in the DBA/2 strain. Five overlapping fragments of the AhR coding sequence were obtained from liver RNA by reverse transcriptase synthesis of a cDNA first strand, followed by polymerase chain reaction amplification of these cDNA sequences (RT-PCR). Analysis by HF and SSCP revealed the presence of sequence differences in three of the five fragments. When the complete nucleotide sequence of the coding regions was determined by PCR sequencing, we found a total of ten nucleotide differences between the two alleles, nine of which localized to the three fragments where differences were detected by HF and SSCP. Five of the differences are silent. Of the other five, one changes the opal termination codon in Ahrb-l to the codon for Arg in Ahrd, extending translation of the mRNA by 43 amino acids and accounting for the larger size of the AhR peptide in DBA/2 mice. One of the four remaining differences causes the replacement of a leucine residue in Ahrb-l by a proline residue in Ahrd, and breaks a potential alpha-helix near the AhR Q-rich region; it is likely that structural changes associated with this amino acid change are responsible for the differences in agonist affinity observed between the Ah receptors of these two strains of mice.

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