A large variety of environmental pollutants are known to be toxic and/or carcinogenic. It is therefore important to understand how organisms respond to these xenobiotics at the cellular and molecular levels. A major element in the detoxification of these substances is the induction of both the cytochromes P45O1A1 and 1A2, as well as a variety of additional proteins, which serve to oxidize these nonpolar compounds. Paradoxically, instead of converting some of these pollutants to harmless forms, these proteins can often activate their carcinogenic potential. The goal of this proposal is to determine the molecular basis of the regulation of a set of genes which is induced by polycyclic hydrocarbons in mice. First, we plan to continue our work defining the novel post- transcriptional mechanism of induction of the P4501A2 gene. We have already shown that the gene is induced at the level of nuclear mRNA processing in a hydrocarbon receptor-dependent manner. While regulation of nuclear processing has been postulated for many years, there is no direct evidence to date that such regulation is used for any specific gene. The cytochrome P4501A2 gene may allow the identification and characterization of the cis- and trans- elements that are required for hydrocarbon- dependent post-transcriptional regulation. Second, we plan to apply the recently developed technology of normalized cDNA libraries, to identify a larger set of carcinogen-inducible genes, and to identify their level of regulation. Specifically, we will construct normalized cDNA libraries corresponding to mRNA expressed in hydrocarbon treated mouse liver, and we will screen the libraries using a combination of subtractive hybridization methods and direct plus-minus screening techniques. This approach will permit the identification of relatively low abundance inducible genes over the background of abundant liver-specific and cytochrome P450 clones. Straightforward analysis by northern hybridization and nuclear run-on transcription will permit an assessment of the levels of gene control operating on each gene. By comparing a set of genes which respond to the same hydrocarbon inducer, it will be possible to better understand the diversity of-mechanisms controlling hydrocarbon gene control.
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