The large intron of the κ immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the κ gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as λproducing cells, and have demonstrated by DNAae footprint analysis fullS protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DM5 reac tivity of DNA in the murine 70Z/3 cell line after it is induced to κ expression. These alterations occur at gu.anine residues which are part of the 11 bp core sequence, and are identical to those observed in cells constitutively expressing κ. This provides direct evidence for the induced binding of the tissue specific factor to intact chroniatin. In intact chromatin we also observed significant alteration in the reactivity of a gua nine, 3′ of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.
Bibliographical noteFunding Information:
This work was supported by Grant GM 37687 from the National Institutes of Health to BVN, with additional support fron the Searle Scholar Program, and a Diabetes-Endocrinology Research Center Grant to the University of Iowa. D.L. received support from V.A. Medical Research Funds: R.H. received support from a thrombosis-hemostasls fellowship from the National Institutes of Health; G.S. received support from a grant from the National Institutes of Health.