This program is designed to develop as complete as possible an understanding of the control of transcription of certain genes in lymphoid cells and in a lymphocyte-specific virus. The topics to be investigated are the following questions: 1. What determines the B lymphocyte-specific transcriptional activity of immunoglobulin genes?; 2. How is transcription of T cell receptor (TCR) genes controlled?; 3. What controls IL-2 induction in T lymphocytes?; 4. What controls the induction and repression of terminal deoxynucleotidyl transferase in early lymphoid cells?; 5. What controls induction of human immunodeficiency virus in T lymphocytes?; 6. What is the mechanism of action of the tat gene system in human immunodeficiency virus? Characterization of transcriptional control for these various systems involves, in many cases, the same set of experimental activities. We first use a transient transfection protocol with various cell types to demonstrate that the cloned gene shows transcriptional control. This can be done with the intact gene or with fragments connected to a reporter gene (usually chloramphenicol acetyl transferase). Then deletion analysis can grossly demarcate the important regions. Electrophoretic mobility shift assays with regions to which putative regulatory proteins can bind allow a fine mapping binding sites. Point mutations in these binding sites then show if the sites have regulatory significance. Purification of the proteins followed by cloning can then allow further physical and functional study, especially using in vitro transcriptional systems.
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