I propose to investigate the molecular basis of a hereditary disease that affects heme biosynthesis in man (porphyria). Several procedures will be utilized to obtain DNA probes of two heme-biosynthetic genes (including a method for cloning complementary DNAs by their ability to genetically complement yeast heme-deficient mutants). These problems will be used in experiments designed to analyze the expression of these genes fibroblast cultures and cell lines of normal and porphyric individuals. In one particular disease, acute intermittent porphyria, patients are heterozygous, having one normal allele and one defective allele; the latter produces no gene product (i.e., no porphobilinogen deaminase, PBGD). Cells from individuals with acute intermittent porphyria will be characterized for deletion of the PBGD gene, deficiencies in transcription of the PBGD gene, processing of PBGD nuclear RNA, PBGD mRNA half-life, and mRNA translation. The techniques that will be used include: pulse-chase RNA labeling, RNA blotting (Northern blots), S1-nuclease transcript mapping, in combination with various cell fractionation and nucleic acid hybridization procedures. Once characterized, molecular cloning and DNA sequencing of the mutant allele(s) will provide a precise location of the defect(s). The data obtained from studies on defective genes will be utilized to understand better the regulation of normal gene expression. By taking a cue from these and other mutations known to affect gene expression (such as Beta-thalassemia), in vitro """"""""mutations"""""""" will be constructed in cloned genes to test various hypotheses concerning the nucleotide sequence requirements for RNA splicing, and mRNA stability. Various techniques of introducing these cloned sequences into mammalian cells will be used, and the expression of these altered genes will be studied. Initial experiments will be performed with both naturally occurring and in vitro-constructed mutations of the Beta-globin gene. When cloned genes for heme biosynthetic enzymes become available, these also will be utilized. With this combined approach, these studies should yield information on the molecular defect of a hereditary disease as well as improve our understanding of gene expression.
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