The goal of this grant and its predecessor is to study the molecular events related to heave chain gene expression. The last grant focused on the structure and rearrangement of C/H genes. An in depth characterization of the Mu-C delta locus in mouse and man has provided a framework to address fundamental issues of transcriptional and post-transcriptional regulation. In this renewal, we propose to extend the work on Mu-delta regulation, primarily be exploiting in vitro expression technology, with an eye toward understanding the differential roles of the gene products. Specifically, we will determine the levels at which IgM and IgD are regulated in human B cells; significance draws on the dramatic evolutionary divergence of C delta structure in this species while retaining phenotypic conservation in IgD function. We propose a series of transfection studies aimed at fine dissection of 3' and regulation in the murine mu-delta locus. That is, what is the mechanism(s) governing membrane vs. secreted carboxyl-terminus and/or Mu vs delta exon selection? We propose studies on V/H promoter-associated sequences, focusing on protein: DNA interactions and the regulatory effects of T cells on this interaction. We propose a novel cDNA cloning strategy for characterizing putative transcription termination and promoter-associated factors. We provide preliminary feasibility data and design projects for exploitation of antigen-specific IgM and IgD transfected B cell lines. Specifically, we will investigate the role of phosphorylcholine-specific mIg receptors, both in wild- type and structurally modified forms, in B cell activation, processing, and antigen presentation. Finally, we devise a molecular approach to address the differential roles of mIgM and IgD in neonatal B cell tolerance.
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