The overall objective of the proposed research is to investigate the inborn errors and molecular genetics of the four human cytosolic heme biosynthetic enzymes, delta-aminolevulinic acid dehydratase (ALA-D), hydroxymethylbilane synthase (HMB-S), uroporphyrinogen III synthase (URO-S) and uroporphyrinogen decarboxylase (URO-D). Using the full-length hepatic cDNAs for ALA-D, HMB-S, URO-S and URO-D which have been cloned and sequenced in this laboratory, their respective full-length erythroid cDNAs will be isolated and characterized for identification of tissue-specific sequences in their 5' or 3' regions, determination of their respective cap sites and for prokaryotic expression of the active enzymes. Sub-chromosomal regional localization for HMB- S and URO-S will be assigned by somatic cell and in situ hybridization. Genomic sequences encoding all four enzymes will be isolated; the 5' and 3' regions as well as the intron-exon boundaries will be sequenced and compared. For studies of transcription, chimeric constructs of the genomic 5'-flanking sequences of each gene and the bacterial chloramphenicol acetyltransferase gene will be transfected into K562 erythroid and HepG2 hepatic cells. Deletions and substitution mutations in specific 5' regions and DNase I protection experiments with hepatic and erythroid nuclear extracts will be performed to identify transcriptional regulatory elements and nuclear factors involved in the tissue-specific expression of these genes. The nature of the molecular lesions in unrelated patients with ALA-D deficiency, acute intermittent porphyria (AIP) and cogenital erythropoietic porphyria (CEP) will be determined. Efforts will be directed to identify informative RFLPs for the precise diagnosis of heterozygotes for AIP and porphyria cutanea tarda (PCT. The biochemical and molecular abnormalities in cats with HMB-S dificiency will be characterized. This animal model will permit the unique opportunity to evaluate the metabolic effects of therapeutic modalities, including bone marrow transplantation and retroviral-mediated gene transfer. These studies should provide 1) increased understanding of the structure, organization and tissue-specific regulation of these heme biosynthetic enzymes, 2) delineation of the molecular lesions in these disorders as well as 3) new methods for the precise diagnosis and potential therapy of these inborn errors of heme biosynthess.
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