Polycyclic aromatic hydrocarbons (PAH's) are widespread pollutants. Many are metabolized to highly carcinogenic derivatives. The principal enzyme activity involved is cytochrome P1-450 dependent aryl hydrocarbon hydroxylase (AHH). PAH's, in addition to being substrates, also induce P1-450. Induction is mediated by the Ah receptor. This receptor also mediates pathogenesis (including carcinogenesis) of many environmentally important polychlorinated hydrocarbons, but by an unknown mechanism. P1-450 is highly inducible in mouse Hepa-1 cells. Non-inducible mutants were isolated. A few are dominant. The majority are recessive and fall into four complementation groups. Mutants in genes B, C and D are defective in Ah receptor functioning. Dominant mutants synthesize a repressor of P1-450 transcription. The main objectives of the proposed research are to clone, analyze, and utilize the B, C, D and dominant genes. Human or rat genomic DNA-derived transfectants of individual B-, C- and D- mutants have been obtained in which P1-450 inducibility is restored. Phage or cosmid libraries of secondary or tertiary transfectants will be made, and plaques or clones containing all parts of the B, C, or D genes identified by probing with human or rat repetitive sequences. The dominant gene will be isolated using a similar transfection strategy. Each cloned gene or gene fragment will be utilized to determine i) how many closely related genes exist; ii) whether the corresponding mRNA is induced by PAH's in human lymphocytes, and whether the degree of inducibility in different persons is associated with susceptibility to cigarette-induced lung cancer, and iii) to map the human genes. Portions of each gene will be ligated to an E. coli expression vector, and the corresponding fusion protein isolated and used to generate antisera. Mammalian cells expressing high levels of the """"""""natural"""""""" proteins corresponding to each gene will be obtained by cotransfecting and coamplifying the genes along with a cloned dihydrofolate reductase gene. The proteins will then be isolated from these cells using the above antisera. These proteins (three of which may be structural components of the Ah receptor) will be tested for their ability to bind TCDD, the cloned P1-450 gene, and each other. Finally the AHH regulatory genes or gene fragments will be used to isolate cDNA's and these, and interesting portions of the genes, will be sequenced. These studies therefore promise to give condiderable insight into the structure, role and function of the Ah receptor and the mechanism of regulation of cytochrome P1-450.
Showing the most recent 10 out of 67 publications
