Adenosine deaminase (ADA), an enzyme of the purine salvage pathway, is a ubiquitous enzyme in mammalian tissues. However the levels of expression are regulated in a tissue-specific manner with ADA activity spanning a range of several hundred-fold among various tissues. Expression of ADA is regulated temporally during development of certain tissues and ADA expression is also altered during the functional differentiation of specific cell types. Abnormal levels of expression of ADA are associated with disease states, with very low levels causing severe combined immunodeficiency disease and high levels resulting in hereditary hemolytic anemia. The long-term objective of this application is to define the mechanisms and elements involved in determining the level of tissue- specific ADA expression and eh variation of expression during normal development and differentiation. Recent isolation and characterization of DNA encompassing the ADA gene makes this feasible. The ADA gene contains a basic promoter comprised of G/C rich elements of the type that bind Spl. A unifying theme for regulatory elements that interact with Spl responsive promoters has not been identified tissue-specific pattern.
The specific aims of this application are to define and characterize positive regulatory elements identified in the first intron of the ADA gene, negative elements identified in the 5'-flanking region, and any additional elements in intragenic and specific and development expression of ADA. Primary methods will be construction and transfection of hybrid genes and production of transgenic mice with fragments of these constructions. Assays for gene expression, at the level of enzyme activity and mRNA, will allow evaluation of the significance of particular DNA elements and their interactions in regulation of expression of ADA. In concert deletional analysis, nuclease hypersensitivity, genomic footprinting, and competition studies will map and define the elements. These methods will also be used to determine the fine structure of the G/C rich basic promoter and elucidate its interaction with the defined regulatory elements. Knowledge of the elements necessary to specify proper ADA expression may be critical in therapeutic applications such as gene replacement therapy.
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