Complete assembly of T cell receptor (TCR) variable region genes does not occur in B cells and complete assembly of immunoglobulin (Ig) variable region genes does not occur in T cells. There is mounting evidence that control of this process occurs at the V to DJ rearrangement step. The goal of the project described in this proposal is to identify the cis- and trans-acting control elements of the TCRbeta V to DJ rearrangement step. Cis-acting elements will be identified by mutational analysis of the germline TCRbeta gene. Two systems will be used to determine the ability of various mutants to rearrange. The first involves introduction of the mutants into a B cell tumor which overexpresses the RAG genes, followed by analysis of the recombination products by PCR or Southern blotting. The second involves introduction of these genes into ES cells followed by injection of ES cells into blastocysts of RAG-2-/RAG-2-mice, which are unable to rearrange endogenous TCR and Ig genes and therefore have no mature B and T cells. All of the B and T cells from the resulting somatic chimeric mice will be of ES cell origin and can be easily analyzed for V to DJ rearrangement of the introduced TCRbeta genes by PCR or Southern blotting. Once the cis-acting element has been identified, experiments will be carried out to determine if it defines a protein binding site. If so attempts will be made to isolate and clone the cDNA for the protein. In addition, the role of transcriptional activation and DNA methylation in controlling the V to DJ rearrangement step will be determined. Understanding the control of TCR and Ig V to DJ gene rearrangement should provide insight into the genetic control of lymphoid differentiation and ultimately help us understand abnormalities in this process leading to immune dysfunction and lymphoid malignancies.
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