The immunoglobulin (Ig) genes provide a paradigm to study many interesting biologically relevant problems, which include: DNA sequence organization and evolution, tissue-specific transcriptional regulation, site-specific recombination, somatic hypermutation, regulation of replication timing, modulation of DNA methylation patterns, developmental control of many of these events, allelic exclusion and the relationships between chromatin structure and function. This proposal focuses on the mousse kappa immunoglobulin (Ig) gene locus. Our goals during the proposed project period are to elucidate features of the genomic organization, chromatin structure, and relationship between chromatin structure and function within this locus. Our experimental approach is designed to uncover new cis-acting regulatory elements.
These specific aims are described more fully below: 1. To isolate the entire mouse kappa Ig locus from a genomic library contained within yeast artificial chromosomes (YACs). We will establish contigs, a physical linkage map between selected V gene families, and a low resolution restriction endonuclease map. 2. To locate new putative cis-acting regulatory sequences within the kappa Ig gene locus. We will selectively map endogenous topoisomerase II and exogenous DNase I cleavage sites within the chromatin of various cell lines of B- and non-B-lymphocyte lineages. Special attention will be placed on distal upstream and downstream regions. 3. To functionally elucidate cis-acting regulatory sequences within the kappa Ig gene locus. We will create deletions within YACs containing the mouse kappa Ig locus of selected known and putative new cis-acting regulatory sequences (identified in specific aim 2). These YACs containing engineered kappa Ig sequences will be introduced into endogenous-kappa- gene-deficient pre-B cells for germ-line transcription and recombination assays, and into the germline of endogenous-kappa-gene-deficient mice for a variety of functional assays. In summary, these studies have the potential of identifying novel regulatory elements, which may include locus control region(s) (LCRs), domain boundary sequences, and components yet to be understood that may further regulate the efficiency of the immune response.
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