There is much evidence that inherited variations in catecholamine metabolism play a role in certain neurologic disorders. A long term goal in this research proposal is to elucidate the molecular genetic basis of inherited variations of human catechol-O-methyltransferase (COMT, EC 2.1.1.6), a key enzyme in the metabolism of catecholamine neurotransmitters. Proposed here is to examine inherited variations in COMT activity levels as determined by variations in DNA nucleotide sequence within selected families (those segregating """"""""high"""""""" or """"""""low"""""""" activity levels). Studies will initially be directed toward the cloning of cDNAs for human COMT by screening of a human cDNA library with a cDNA probe for either rat or bovine COMT (eight putative positive clones are on hand, which must first be verified by at least one of the methods outlined in this application). If none of these are confirmed, alternative strategies will include, rescreening bovine and rat cDNA libraries with a labelled cDNA probe made from immunopurified COMT mRNA already on hand, or with oligonucleotide probes made according to COMT-peptide amino acid sequences (which have yet to be determined). cDNAs will be characterized by Northern blotting, hybrid selection, in situ hybridization and nucleotide sequencing. They will then be used to isolate clones from screening a human genomic library. The genomic clones will be characterized by restriction mapping, nucleotide sequencing, and S-1 mapping to determine the intron and exon positions. Chromosomal mapping of COMT genes will be done by in situ hybridization to human metaphase chromosomes. Inherited variations in erythrocyte COMT activity will be examined by linkage analysis of genomic DNA from families segregating high or low activity levels with the use of genomic and cDNA probes for COMT. DNA from identified families will be analyzed by cleavage with specific restriction endonucleases, resolution on agarose gels, genomic blotting and hybridization to the probes. Molecular genetic methods will help determine if sequence variations within or near the COMT gene are the basis for variations in erythrocyte COMT activity, as well as help clarify how multiple forms of the COMT enzyme (soluble and particulate) relate to each other; that is, do they arise from the same gene or from different genes, and how can this information help explain functional differences.
