In this renewal application, the cloning of the rat 3a- HSD/DD gene will be completed. Sequence upstream (-4.0 kb) to the strong distal enhancer will be obtained. Nested deletions of existing pCAT constructs will locate this enhancer within a 200 bp fragment. Trans-acting factors that bind to the distal enhancer will be identified by band-shift and DNase-I footprinting. Oct-factor consensus sequences are uniquely located in the NRE. Several approaches will be used to determine whether Oct - factors are repressors of the 3a-HSD/DD gene: 1) gel-retardation assays and DNase-I footprinting will determine whether nucleoproteins and recombinant Oct bind to octamer consensus sequences within the NRE; 2) site-directed mutagenesis of the Oct-sites will be used to restore CAT activity in reporter gene constructs containing the NRE; and 3) p delta NRE-CAT vectors (which contain -498 to +61 bp of the 3a-HSD/DD gene) will be co- transfected with pNRE (-799 to -498 bp) to see if pNRE-Oct-1 complexes will act in transit to suppress CAT activity. Primary cultures of rat hepatocytes will be used to study the mechanisms by which glucocorticoids increase 3a-HSD/DD gene expression. The ability of steroid hormones to regulate the 3a-HSD/DD gene will be related to the structure/function of the SRU. Typically, Dex. will be used to stimulate CAT activity in HepG2 cells co-transfected with pSRU-hsd-CAT (where hsd is the basal promotor) and pRSV-hGR (where the human glucocorticoid receptor is linked to the constitutive RSV promotor). Co-transfection paradigms will investigate synergy between steroid hormone receptors and AP-1 factors in the trans-activation of the SRU. Mutagenesis of the SRU will identify specific cis-elements involved in gene regulation. Positive findings would implicate steroid hormones as elicitors of the complete carcinogenic potential of PAHs.
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