The long term objective of this research is to investigate the molecular mechanism for the generation of the diversities of immune receptors. The V, (D), and J segments of the Ig and TCR genes are assembled by somatic recombination during the lymphoid differentiation. These recombination processes are mediated by the recombination signal sequences (Rss) and the V(D)J recombinase. To elucidate the molecular mechanisms of the V(D)J recombination, it is essential to characterize lymphoid-specific proteins that bind the Rss. We have cloned the cDNA for a protein, Rc, which is predominantly expressed in thymocytes by the ability of Rc to bind the Rss. Subsequently, we have shown that Rc binds the kappa-B motif of the Ig kappa chain gene enhancer as well. Significantly, fusion proteins for Rc have a tendency to form multimeric structures and to cleave DNA substrates at sites proximal to the Rss. These activities are relevant for the V(D)J recombination. Another interesting property of Rc is that Rc inhibits the activity of a transcription factor, VP16, in an """"""""effector-reporter"""""""" assay. Therefore, we hypothesize that Rc is a regulator for the transcription of the Ig kappa light chain gene and for V(D)J recombination. Rc may modulate V(D)J recombination by modifying the accessibility of the Rss via its possible involvement in the transcription of Ig gene segments. Thus, Rc may provide a link between gene rearrangement and transcription. The first specific aim of this proposal is to investigate the role of Rc on transcription of reporter genes in lymphoid and non-lymphoid cells. The effect of the GAL-Rc fusion protein or Rc alone on the transcription of a gene controlled by the yeast GAL4 binding site, or by the kappa-B motif of an Ig light chain gene, respectively, will be evaluated. The second specific aim is to investigate the role of Rc in the recombinase system. Correlation between the expression level of Rc and the recombination frequency will be investigated. Rearrangements for plasmid recombination substrates and for endogenous Ig kappa chain gene segments will be examined. The third specific aim is to investigate and characterize the molecular properties of Rc and to identify its possible associated protein(s). Fusion proteins of different sizes will be produced to map the DNA binding domain(s) and the transcription activation/inhibition domain(s) of the Rc protein. Antibodies for Rc will be generated to define the tissue distribution, cellular localization, and state of phosphorylation of the endogenous Rc protein. Proteins that associated with Rc which may be components for the V(D)J recombinase will be identified. V(D)J recombination is the main contributing factor for the diverse specificities of Ig and TCR molecules. A lesion in the specificity of the V(D)J recombinase could lead to aberrant gene rearrangements, or chromosomal translocations. These events often result in tumorigenesis. Research on the molecular mechanism of V(D)J recombination is, therefore, important to understand the generation of immune diversity, and may shed light to prevention of tumorigenesis due to errors of the V(D)J recombinase.
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