The long term objective of this proposal is to study the structure, function and regulation of insect cytochrome P450 genes and proteins. P450 monooxygenases are involved in the metabolism of endogenous substrates. P450 enzymes also serve to detoxify foreign chemicals of natural or synthetic origin. In the latter case, overexpression of P450 genes can lead to insecticide resistance. Moreover, the induction of P450 enzymes by phenobarbital and by many other types of chemicals in insects may provide a useful model for similar induction in vertebrates. The following specific aims will be addressed: 1. To clone and sequence the genes and reconstitute activity of the P450 proteins responsible for essential physiological functions. 2. To study the role of cytochrome b5 and the stoichiometry of P450 reactions in reconstituted systems. A house fly cytochrome b5 cDNA will be expressed in E.coli and the purified protein used in reconstitution studies with house fly P450 and NADPH cyt P450 reductase that have been expressed and purified. The coupling of insect P450 reactions will be studied. NADPH consumption and oxygen consumption will be measured in conjunction with the conversion of substrate to product and the generation of superoxide and hydrogen peroxide. 3. To study the mechanism of induction of P450 genes by phenobarbital using Drosophila as model. Transient expression of reporter genes in transfected Drosophila cells under the control of the 5' flanking region of the phenobarbital-inducible CYP6A2 gene will be studied. P-element vectors carrying the 5' flanking region of the CYP6A2 gene and a reporter gene will be used to transform Drosophila. Transgenic flies will be characterized with respect to their response to phenobarbital treatment in vivo. EMS mutants with altered phenobarbital induction phenotype will be generated and characterized. These studies should provide a useful model for the mode of action of this class of P450 inducers.
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