Our laboratory has recently cloned and identified human cDNA clones that encode their equivalents to rabbit cytochrome P-450 1, the constituitive extra-adrenal 21-hydroxylase, and the TCDD inducible P-450 4 and P-450 6. This proposal outlines a series of experiments that will be undertaken to obtain information concerning their structure, an understanding of their function and the mechanisms which underly their differential regulation at the molecular level. Following the isolation and characterization of full length clones for the human P-450s 1, 4 and 6, these clones will be stably transfected into cells under the control of active eucaryotic promoters. The integration of these transcriptionally active cDNAs in human cells under conditions where there is no constitutive expression of these P-450s, will allow us the unique opportunity to study their independent catalytic activities. With the use of monoclonal antibodies directed to the rabbit P-450s, it may also be possible to use these reagents as tools to delineate the substrate specificity of the clonal P-450 activities. The P-450 structural genes will be identified and important 5' sequences will be used in DNA transfection experiments to identify the structural regions involved in the association of what may be transacting components that participate in eliciting the differential expression of these P-450s. In conjunction with these experiments, the P-450 genes will be characterized for intron/exon structure with careful comparison of the cDNAs and the gene exon sequences. This will be particularly important for P-450 1, since Southern blot analysis suggests P-450 1 is part of a unique multigene family. Selected sequences, either from the cDNAs or genomic clones will be used to determine the tissue specificity of the P-450s from a human tissue bank that has been developed at UCSD. Combined, we anticipate that a clear understanding of the function, regulation and tissue specificity in humans of these important enzymes will evolve from these studies.
