A goal of this laboratory's efforts is to employ molecular genetics to understand the structure, regulation, and function of specific human genes that are expressed in hematopoietic cells and relevant to disease. An important host defense system of phagocytes is a plasma membrane associated NADPH oxidase that reduces molecular oxygen to superoxide. The components of the oxidase system, their correspondence with specific genetic loci, and the primary protein defect(s) in chronic granulomatous disease (CGD) (a spectrum of genetic disorders that incapacitates this enzyme system) have been largely unresolved. Recently, the gene and cDNA for the product lacking in X-linked CGD, the classical inherited disease in the system, was cloned by """"""""reverse genetics"""""""" and shown to encode a 90 kd membrane glycoprotein subunit of the phagocyte b-cytochrome. In the proposed research the structure, expression, and function of the 90 kd X-CGD protein will be pursued from a molecular perspective. Attention will be directed toward delineating the cis-acting DNA elements that regulate lineage specific (phagocytic) expression of the gene by gene transfer of promoter/enhancer fusions and toward identification and characterization of nuclear DNA-binding proteins that interact with the critical DNA regions. To study the X-CGD protein and its function antibodies to specific domains will be generated to determine its membrane orientation and to modulate the activity of the system form the extracellular space. A functional assay for the protein will be established by transfer of full-length cDNA into heterologous phagocytic and non-phagocytic cell lines, and into deficient myelomonocytic cells. With this assay and in vitro mutagenesis of the cDNA the role of specific domains of the protein and interaction with a 22 kd component of the phagocyte b-cytochrome will be approached directly. To study the interaction of the 90 kd and 22 kd components, cDNA for the 22 kd will be isolated and used in coexpression experiments. Finally, the genetics of specific X-CGD mutations will be investigated, especially in variant patients with residual b-cytochrome activity, and RFLP probe suitable for improved prenatal diagnosis of X- CGD by DNA analysis will be developed.
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