The long-term goal of the proposed research is to elucidate the structure and molecular mechanisms controlling expression of terminal deoxynucleotidyl transferase (TdT) during hemopoietic cell differentiation. TdT is a DNA polymerase which catalyzes polymerization of deoxynucleoside triphosphates without template instruction. Enzyme activity is expressed in several precursors of the lymphocyte series and in leukemic counterparts of these cells. Our experimental appraoch is to study the structure of homogeneous forms of the enzyme we have isolated and to use recombinant DNA procedures to prepare molecular probes for the TdT gene. In previous studies we have correlated the appearance of TdT activity and antigen with a variety of human leukemic cell types. We have purified three molecular forms of TdT from leukemic cells and from thymus. We have prepared antibody and have studied antigenic relatedness of TdTs. We now wish to determine the origin of these different forms of human TdT and to prepare molecular probes which can be used to determine how expression of the TdT gene is modulated during development and neoplastic transformation. Our specific aims are 1) to use homogeneous TdT proteins we have isolated for detailed structural studies, 2) to use poly(A) + RNA enriched for TdT sequences to isolate the TdT gene(s) and probe for possible mechanisms of tissue specific gene expression and 3) to complete a series of biological studies aimed at studying TdT during evoluation, ontogeny and differentiation. We believe that these studies will lead to a better understanding of TdT, a versatile, but enigmatic enzyme which is of enormous importance as a biochemical reagent, as a marker of early T-cell development and as a clinical tool in the differential diagnosis of leukemia. Isolation of homogeneous TdT renders feasible detailed studies of protein structure. Access to large quantities of human tissue with very high levels of TdT specific mRNA and to monospecific antibody to TdT is pivotal for studies of synthesis, processing and regulation of TdT in human cells.