Antithrombin III (ATIII), a glycoprotein with Mr of about 60,000 plays a critical role in hemostatic regulation. Congenital deficiency of ATIII is a common disorder of hypercoagulability which in some cases results from the inheritance of an apparently mutant ATIII gene. In this proposal, the methods of recombinant DNA technology will be used to construct a detailed restriction map of the normal human ATIII gene. Common DNA restriction enzyme polymorphisms will then be used to identify and isolate abnormal ATIII genes from genomic DNA libraries of ATIII-deficient individuals. DNA sequencing of selected regions of these genes should allow for the identification at the nucleotide level of the specific lesion(s) responsible for this disorder. In additional experiments, an ATIII """"""""minigene"""""""" will be constructed by selective elimination of all but one of the intervening sequences of the normal gene. The size of the minigene will be only about 20% that of the native gene, thus facilitating subsequent manipulations. The minigene will be propagated in a eukaryotic vector which allows for extremely high levels of ATIII production. In vitro mutagenesis of the minegene in this vector will permit a study of how single amino acid substitutions affect various ATIII functions. Additional mutations in the 3 feet-untranslated region of the ATIII gene will be introduced to determine the sequence requirements for polyadenylation, a ubiquitous but little understood eukaryotic function.
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