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Nucleic Acids Research, 1988, Vol. 16, No. 3 953-967
© 1988

Articles

Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions

Robert Hromas, Urs Pauli2, Adriana Marcuzzi, Dave Lafrenz1, Harry Nick2, Janet Stein2, Gary Stein2 and Brian Van Ness*

Department of Biochemistry, University of Iowa Iowa City, IA 52242 1V.A. Medical Center, University of Iowa Iowa City, IA 52242 2Department of Biochemistry, University of Florida Gainesville, FL 32610, USA

*To whom correspondence should be addressed at: Institute for Human Genetics, 5-110 Moos Tower, 515 Delaware St., SE., Box 206 UMHC, University of Minnesota, Minneapolis, MN 55455, USA

Received October 16, 1987. Accepted January 7, 1988.

The large intron of the {kappa} 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 {kappa} gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as {lambda}producing 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 {kappa} 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 {kappa}. 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.


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