The proposed experiments are designed to investigate how the mammalian adenosine deaminase (ADA) gene can be regulated in a tissue-specific manner. Information on how differential gene regulation can be achieved in different cells of higher eukaryotes is essential for understanding how specialized cells of multicellular organisms can arise from unicellular fertilized eggs via specific developmental genetic programming. This knowledge will undoubtedly provide insight into why cancerous cells can deviate from their normal developmental program and grow out of control. There is, currently, also considerable clinical interest in the mammalian ADA gene. For a number of reasons, the ADA gene is considered the prime candidate as a model system to attempt gene replacement therapy in man. Given the complex tissue-specific regulatory aspects of the ADA gene, a clear understanding of its tissue-specific regulation may be necessary for such studies to proceed successfully.
The specific aims of this proposal and the general approaches proposed to achieve them are listed as follows: I. Determination of whether tissue-specific ADA gene regulation is achieved by regulation at the mRNA or protein level. This will be accomplished by quantitating ADA protein and mRNA steady-state levels and synthetic rates in different human cell lines which show tissue-specific regulation. II. Cis-regulatory elements which confer tissue-specific regulation to the ADA structural gene will be identified, isolated and characterized by functional gene transfer assays. III. DNA sequences responsible for generating trans-acting factors which determine tissue-specific ADA gene regulation will be identified and isolated by DNA-mediated gene transfer approaches and characterized. IV. The conformation and organization of the ADA structural gene in mammalian chromatin will be characterized. The relation of structure to function will be investigated through the analysis of DNA methylation effects and sequence-specific nucleosome formation on the ADA structural gene.
