The broad, long-term objective in this proposal is to gain insight into the mechanism by which phenobarbital and phenobarbital-type (e.g. other barbiturates, PCBs with ortho chlorines, DDT, phenytoin, 5,5- diphenylhydantoin) hepatic cytochrome P450 inducers control gene expression by using a responsive (rat) and non-responsive model (rainbow trout) to identify gene regions which mediate the induction response. The non- responsiveness of trout is similar to the non-responsiveness of humans and elucidation of the molecular biology of the cytochrome P450 gene superfamily of rainbow trout, along with comparison to responsive mammalian systems, such as the rat, should provide insight into the phenobarbital induction mechanism as well as providing for the further development of rainbow trout as a lower vertebrate model of mammalian drug metabolism. In order to accomplish this goal it is intended to test the hypothesis that rainbow trout liver contains genes belonging to the P450 gene superfamily in addition to the already described P450IA1 gene and that the rainbow trout model developed can be used to gain insight into the molecular biology of mammalian P450 genes.
The specific aims i n this proposal are a) to identify and begin to characterize the DNA within the trout genome which has sequence similarity to the phenobarbital-inducible P450 (P450IIB1) gene of the rat including the identification of possible regulatory regions, coupled with comparison of the sequences of appropriate, newly obtained, cDNAs with published mammalian and other P450 sequences; b) identification of P450 genes in addition to P450IA1 in trout by development of a complementary DNA (cDNA) probe corresponding to the highly-conserved P450 heme-binding region (P450hbr cDNA), by the use of subtraction cDNA libraries, through the use of a cDNA probe corresponding to P450IIB1 of the rat, and also by the use of available antibodies to screen expression libraries; c) identification and characterization of the upstream regulatory regions of the trout P450IA1 gene through construction of an extended cDNA clone of the P450IA1 pfP1 450-3' cDNA of rainbow trout and synthesis of oligonucleotides corresponding to published regulatory region sequences. These studies will have broad application in the areas of gene expression regulation in eucaryotes, mechanisms of signal transduction, the regulation of gene expression during development, the evolutionary divergence of the P450 gene superfamily, and in the study of the fate of chemicals in aquatic species consumed by man.
