The overall aim of this program is to gain as complete a knowledge as possible of the events that transpire during the differentiation of B lymphocytes and to begin to extend the analysis to the nervous system. This work is particularly relevant to the study of autoimmune disease, immunologic responses, immunologic memory and neuronal deficits.
The specific aims of the next granting period are to understand the role of a single transcriptional enhancer, the intron enhancer of the Kappa (K) light chain; to understand the immunoglobulin gene rearrangement process and its control at a biochemical level; to develop a method to mark memory cells and to extend the methods of analysis of immunodifferentiation to the study of neuronal differentiation. Kappa gene expression is thought to be controlled by 2 enhancers. The various roles of these enhancers in immunodifferentiation will be analyzed using homologous recombination to make critical mutants in ES cells that can then be used to reconstitute the lymphoid systems of RAG-2-deleted mice. ES cell homologous recombination will also be used to insert K regulatory elements into the l genes to examine whether the K/l light chain ratio in the mouse is a consequence of the relative strengths of the transcriptional regulatory elements of the two light chain genes. The immunoglobulin gene rearrangement process is a unique process of DNA rearrangement. To study it, the various relevant proteins will be purified and their in vitro activities will be examined. This analysis will be extended to chromatin and methylated DNA to attempt to understand the various levels of control of the rearrangement process. Analysis in cells will be used to uncover more complex levels of control. There is no unambiguous marker on the surface of memory B and T cells. Transgenic mice will be generated in which the memory B and T cells will be genetically marked and thus can be identified and examined unambiguously. The study of immunodifferentiation has benefitted from the availability of clonal cell lines and methods of chimeric analysis. To extend these benefits to the study of neuronal differentiation, methods will be developed to prepare clonal derivatives of differentiated neurons using oncogenes and a precise labeling procedure. Also to study the role of otherwise lethal genes in neuronal development, a method of chimeric analysis will be developed. It will be applied particularly to analyzing the role of the NF-KB transcription factor in nervous system signaling.
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