Inherited deficiency of adenosine deaminase (ADA) causes a fatal childhood illness, severe combined immunodeficiency disease. Underlying the immunodeficiency are profound disturbances of adenosine and deoxyadenosine metabolism. Abnormal metabolites that accumulate in ADA pattern of expression. In humans there is very high level expression in thymocytes, much lower in mature T-cells, and generally lower yet in most other tissues. One exception, for example, is the mucosa of stomach and duodenum that also exhibits a level of expression comparable to thymus. The rate at which the ADA gene is transcribed varies in a cell-type specific fashion, and closely parallels ADA activity levels. The first intron (15 kb) contains a complex array of cis-active elements that determine, at least in part, the rate at which the ADA gene is transcribed. Only one sub-region of the first intron appears to contain T-cell specific enhancer activity. Since data from bone-marrow transplant-corrected ADA deficient patients indicate that lymphoid-reconstitution alone corrects the immunodeficiency, it is important to determine the molecular mechanisms that underly high level ADA expression in thymocytes. Our basic hypothesis is that one or more specific DNA sequences (core enhancer elements) in the first intron of the human ADA gene interact with cell-type specific DNA binding proteins to regulate ADA expression during differentiation of thymocytes and T- lymphocytes. Testing of this hypothesis will be the major objective of the proposed work and will employ the following specific aims: (1) characterize the cis-regulatory elements(s) within the first intron of he human ADA gene that act as specific enhancers of transcription in thymocytes and T-lymphocytes; (2) purify and characterize thymic protein(s) that bind to the core enhancer element(s) and assess their involvement in regulating the developmental expression of the human gene during T-lymphoid differentiation; and (3) clone and functionally characterize the cDNA(s) and gene(s) that encode the human thymic enhancer binding protein(s). Assignment of functions to putative core enhancer elements and specific enhancer binding protein(s) will require extensive use of transgenic mice and transfection/transient expression assay in cell culture. The expected outcome is an understanding of the molecular mechanism of regulation of ADA expression in human T-lymphocytes and their precursors.
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