Cells of the immune system act in concert to protect against infectious agents and transformed cells. At the heart of this protective system are the clonotypic antigen receptor molecules found on B and T lymphocytes, the immunoglobulin (Ig) and the T cell receptor (TCR). The millions of different genes needed to encode these receptors are assembled from component gene segments by a site-specific recombination process known as V(D)J recombination. Aberrant V(D)J recombination has been linked to human hematopoietic malignancy, and defects in or deregulation of the recombination process could lead to immunodeficiency or autoimmunity. To understand the mechanisms by which V(D)J recombination causes disease, the process needs to be understood at the molecular level. The primary objective of the research described in this application is to identify the enzymatic machinery that carries out V(D)J recombination and determine the role of each enzymatic component in the reaction. Two recombination activating genes, RAG-1 and RAG-2, have been isolated and demonstrated to be necessary and sufficient to activate the V(D)J recombination machinery in non-lymphoid cells. These genes likely encode the critical lymphoid-specific components of the recombination enzyme. They offer unique reagents with which to study the biochemistry of V(D)J recombination. The cloned RAG-1 and RAG-2 cDNAs will be used to direct the expression of the RAG-1 and RAG-2 proteins (and small portions of the proteins) in bacterial and mammalian tissue culture cells. The proteins will be purified by taking advantage of a variety of affinity purification schemes, and used as immunogens to generate polyclonal antisera and monoclonal antibodies specific for the RAG proteins. These immunological reagents will be used to determine the in vivo pattern of expression of the RAG-1 and RAG-2 proteins, both in lymphoid organs and tissues, and in the central nervous system (where the RAG-1 mRNA transcript is found). The antibodies will also be used to identify other components of the V(D)J recombinational machinery by virtue of their interactions with the RAG proteins. The biological activity of the purified RAG proteins will be assayed by introducing the proteins directly into tissue culture cells. The proteins will also be analyzed in detail for their ability to bind DNA (particularly to sequences known to be required for V(D)J recombination) and for their enzymatic activities, particularly as topoisomerases, endo- and exonucleases, and ligases. The information gained in these studies will be used to develop an in vitro V(D)J recombination assay with which the enzymatic mechanism of the reaction can be determined. Identification of the components of the enzyme and an understanding of the role each plays in the recombination reaction should provide insights into the mechanisms by which defects in the reaction lead to human disease.