Diagnostic approaches for the detection of individuals at risk for genetically-predisposed and environmentally-induced diseases (such as cancers and birth defects) are increasingly dependent upon techniques which evaluate DNA. Many endogenous, as well as environmental chemicals are metabolized by a variety of biotransformation enzyme systems, and highly reactive epoxide intermediates have been implicated as toxic products in these pathways. Microsomal epoxide hydrolase (EH)< is a key epoxide- detoxication enzyme, in mammalian cells. The focus of this research program is first to identify, and then to characterize the effects of, polymorphisms associated human EH. To facilitate these analyses, we have isolated human EH cDNA and genomic clones, and have determined that the gene exists in single copy per haploid genome. Our investigations will concentrate on identifying frequently occurring nucleotide alterations within the human EH genes. The presence of variant sequences in the coding portions and 5'-flanking regions of the EH gene will be assessed in human recognition sites, 2) ribonuclease protection analyses of synthetic cRNA:genomic DNA duplexes, and, 3) differential hybridization of oligomer probes to defined regions of the EH gene. Each procedure enables detection of single base differences in gene structure. Regions of polymorphic EH genes will be sequenced following in vitro amplification. To examine the consequences of variant gene structure, site-specific mutagenesis experiments will be performed. Mutagenized EH cDNA templates will be expressed in yeast for the purpose of assessing EH activity profiles of the altered enzymes. DNA substitutions in 5'-flanking regions of the EH gen will be analyzed in transient cell expression assays for their capacities to produce measurable change sin transcription of a chloramphenicol acetyl transferase """"""""reporter"""""""" gene. In summary, these investigations will enable the elucidation and functional assessment of genetic polymorphism in human EH, and should enhance the development of diagnostic procedures allowing identification of individuals at increased risk to chemically-induced disorders.
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