This is a revised application submitted by Dr. Susan M. Hoffman of Miami University (Ohio) to study the evolutionary and functional organization of a cytochrome P450 gene cluster in mice. This project is part of an ongoing study of mammalian genes in the cytochrome P450 superfamily, with the long term goals of achieving a better understanding of both the evolutionary processes that create gene families in general, and the regulatory elements of some P450 genes in particular. This study also represents an initial attempt to use genomic similarities to help assess the suitability of rodent models of P450 activity. Cytochrome P450 proteins are the single most important group of enzymes in the metabolism of exogenous compounds in mammals, and genetic variation in their activity and regulation is known to be medically important. There are between 60 and 200 members of mixed-function oxygenases in the P450 superfamily. There are 14 P450 families, however Dr. Hoffman will concentrate on the CYP2 family, which exists on mouse chromosome 7 (syntenic to human chromosome 19) and has subfamily members CYP2a, CYP2b, and CYP2f. Dr. Hoffman was intimately involved in the detailed characterization of the human cluster and will use this information as the foundation to analyze her forthcoming data in order to better understand the molecular mechanisms responsible for the development of this P450 family and to detect conserved sequences involved in regulating their expression. These goals will be obtained by accomplishing the following specific aims:
The first aim will test the hypothesis that the three gene subfamilies are organized in the same unusual pattern found in humans. A detailed physical map of the gene cluster will be created by restriction mapping genomic clones, and the genes will be localized on the map by hybridization with specific probes. Two large mouse BAC clones have been isolated and a set of 5 restriction enzymes are being use to develop the map. Human and mouse probes will be used to place individual loci on the restriction map and to identify the subfamily membership of each gene.
The second aim will test the hypothesis that there are additional unknown genes and/or psuedogenes within the mouse P450 chromosome 7 cluster. Individual loci corresponding to known sequences will be identified, and previously unanalyzed genes will be sequenced, including their upstream control elements. PCR amplification of diagnostic exons and their sequencing will be used to identify which loci on the map correspond to known mouse cDNA or genomic sequences. Known transcripts will be assigned to loci to determine which are allelic, rather than products of different loci. For the third aim, the human and mouse maps will be compared and potentially orthologous genes will be aligned. Upstream untranslated sequences will be aligned and scanned for conserved elements. This comparison will allow the identification of which genes are ancestral, which are uniquely derived in the two species, and thus Dr. Hoffman will infer the series of molecular events that created the two arrangements.
This aim will be a test for various evolutionary hypotheses. The fourth and final aim will be to analyze any full-length genes that are not associated with known transcripts by RT- PCR in order to determine if they are expressed in various tissues of the mouse.
| Du, Liping; Hoffman, Susan M G; Keeney, Diane S (2004) Epidermal CYP2 family cytochromes P450. Toxicol Appl Pharmacol 195:278-87 |
| Wang, Haoyi; Donley, Kyle M; Keeney, Diane S et al. (2003) Organization and evolution of the Cyp2 gene cluster on mouse chromosome 7, and comparison with the syntenic human cluster. Environ Health Perspect 111:1835-42 |
| Hoffman, Susan M G; Keeney, Diane S (2002) Fine-scale mapping of CYP gene clusters: an example from human CYP4 family. Methods Enzymol 357:36-44 |
| Chen, Ning; Whitehead, Sarah E; Caillat, Alexandre W et al. (2002) Identification and cross-species comparisons of CYP2F subfamily genes in mammals. Mutat Res 499:155-61 |
| Hoffman, S M; Nelson, D R; Keeney, D S (2001) Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11:687-98 |
