The goal of the proposed research is to understand the mechanisms of how genes are regulated in different tissues. This will be accomplished through the study of the human gene encoding debranching enzyme and molecular analysis of Type III glycogen storage disease. In this disease there are patients who lack debranching enzyme activity in both liver and muscle, and patients with enzyme deficiency confined to liver; yet the enzyme is a monomeric protein and appears to be identical in all tissues. The proposed research hypothesizes that debranching enzyme in liver and muscle is encoded by a single gene; its expression, however, is under separate genetic control. Patients with various clinical and enzymatic subgroups of Type III glycogen storage disease are being followed at Duke Medical Center; fibroblasts from these patients have been collected and stored. The debranching enzyme has been purified from porcine muscle and the antibody characterized. The cDNA coding for debranching enzyme from both human liver and muscle will be isolated by screening lambda gtll human cDNA libraries with antibody against porcine debranching enzyme. The debranching enzyme mRNA in these two tissues will be compared by Northern blot analysis, cDNA sequencing and/or sequencing of mRNA by primer extension. The chromosomal gene(s) coding for debrancher enzyme will be isolated, and the molecular structure and number of copies of the genes will be determined. Information about the number of debrancher genes and the sequences of the mRNA from liver and muscle will be used to design strategies to investigate the mechanism responsible for control of tissue-specific debrancher gene expression. This may involve sequencing of the 5' flanking region for two possible transcription initiation sites, or in the case of two different mRNA sequences encoded by a single gene, sequencing the regions of the gene that specify the mRNA unique to liver or muscle. The defective chromosomal debrancher genes from patients with disease confined to liver will also be cloned and the mutations characterized. Molecular classification of Type III glycogen storage disease will be performed at the protein, mRNA, and gene levels. These molecular findings will be correlated to the type and severity of clinical phenotypic expression. Information gained on molecular structure of glycogen debrancher gene and human mutants lacking debrancher activity may lead to identifying DNA regions important in the control of tissue-specific gene expression, and may provide a model for mechanisms responsible for clinical variability in other genetic disorders.
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