One powerful approach to the study of human biology and disease involves examination of the structure and function of specific human genes that encode well characterized products. Until recently attention focused on those genes active at high levels in specialized cells. With molecular cloning technology, a wider spectrum of human genes may be considered. We propose to study two human genes-that for the X-chromosome encoded enzyme phosphoglycerate kinase (PGK) and that for a coagulation inhibitor antithrombin III (ATIII) - in an attempt to approach important problems relating to dosage compensation of X-chromosome genes, the evolution of homologous genes within a family, and and the molecular basis of inherited disease. We will isolate by cloning and fully characterize the human X-chromosome PGK gene in preparation for detailed comparative analyses of the structure and modification of this gene in cells carrying active, inactive, and chemically reactivated X-chromosomes. Our research is founded on our conviction that molecular study of a specific X-chromosome gene whose structure is defined and whose expression can be manipulated offers the most promise for determining potential roles of DNA methylation and chromatin structure in the important and incompletely understood process of dosage compensation. In a parallel project we will isolate and chracterize the cellular gene for ATIII in an effort to distinguish existing models for evolution of a proposed """"""""superfamily"""""""" of protease inhibitors and to determine the molecular basis of ATIII deficiency associated with increased risk of thromboembolism. These latter studies will employ expression of cloned ATIII genes reintroduced into mammalian cells as an assay to identify mutant classes. Due to the moderate size of ATIII protein and its cellular gene, such """"""""functional"""""""" location of mutations, we believe, may be the most practical approach to the identification of underlying genetic lesions, and may serve as an important model for similar analysis of other disorders caused by unknown mutations in moderate-sized human genes.
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