Mechanisms of gene expression control are being investigated for two genes that are active in human B lymphocytes: the immunoglobulin kappa light chain and J chain genes. The expression of the kappa gene is known to be influenced by an enhancer located about 0.l kb 5' of the constant region gene; this corresponds to the position of a B cell-specific DNA base I hypersensitivity site and a segment of about 0.13 kb showing high sequence conservation between mouse, rabbit and man--the Kappa Intron Conserved Region (KICR). We have employed the high-resolution electroblotting technique originally developed for genomic sequence analysis to examine the accessibility of this region to nucleases and to dimethylsulfate (in vivo footprint analysis). The experiments reveal about 0.25 kb segment of DNAase I hypersensitivity and accessibility to restriction endonucleases that extends from the 5' end of the KICR to beyond its 3' end. Two sites of enhanced dimethylsulfare reactivity are located within dyad symmetries at the 5' end of the KICR, consistent with the binding of regulatory proteins to these positions in vivo. We are currently using an exonuclease protection method to detect in vitro the binding of nuclear extract proteins to the same positions and hope eventually to purify such proteins. The human J chain gene was cloned in our laboratory; its expression appears to be regulated coordinatedly with that of immunoglobulin genes in some systems although no sequence homology has been detected between J chain and immunoglobulin genes. Potential regulatory regions of the gene have been studied by two approaches. First, an extensive series of gene constructs have been made to assess enhancer and promoter activities of segments from the gene using transient transfection into B cells. Second, B cell extracts have been found to bind to segments of DNA 5' of the gene in gel retardation assays; the characteristics of this binding are currently being investigated.
