The isolation and characterisation of cytochrome P4504A family genes from the rat.

The phenomenon of induced peroxisome proliferation in rodents has invariably been associated with upregulation of member(s) of the CYP4A subfamily of hemo- proteins. However, regulation of the members of the rat CYP4A family has not been fully examined at the molecular level. Despite the publication of the rat CYP4A1 and CYP4A2 genomic sequences (Kimura et al., 1989a), no functional assays have been performed to determine the mechanism of peroxisome proliferator modulated transcriptional activation of these genes. The objective of this study was to isolate members of the rat CYP4A subfamily of genes and investigate their inducibility by peroxisome proliferators at the molecular level. In this thesis I present results to demonstrate: 1) the isolation and partial characterisation of recombinant DNA clones corresponding to 3 different rat genomic sequences, showing a degree of similarity to the rat CYP4A1 cDNA, 2) identification, by sequencing, of one of the groups of clones (group C) to correspond to CYP4A2 and another (group B) to be, possibly, the genomic sequence of CYP4A3. The identification of the remaining group (group D) remains unknown, but most likely represents a novel member of the rat CYP4A subfamily, 3) the apparent absence of a peroxisome proliferator response element (PPRE) in the 5' flanking DNA sequence of the clones, as determined by hybridisation to the rat acyl-CoA oxidase PPRE oligonucleotide (GTOX285), 4) a lack of a response of a -5.8kb upstream sequence of the newly-isolated human CYP4A11 gene in a co-transfection assay with the mouse PPAR and in the presence of the peroxisome proliferator Wy 14643. The results further extend knowledge on the multiplicity of CYP4A genes in the rat and provide data on the transcriptional regulation of CYP4A genes in rodents and man