TRANSCRIPTIONAL REGULATORY ELEMENTS STIMULATE RECOMBINATION IN EXTRACHROMOSOMAL SUBSTRATES CARRYING IMMUNOGLOBULIN SWITCH-REGION SEQUENCES

We have developed a sensitive genetic assay to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination. Recombination substrates containing mu and gamma-3 chain switch (S)-region sequences, S(mu) and S(gamma-3), are transiently introduced into primary murine B cells cultured with lipopolysaccharide to induce isotype switching. Recombination involving S-region sequences deletes a conditionally lethal marker, the leftward promoter of phage-lambda (lambda-P(L)), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S(mu)-lambda-P(L)-S(gamma-3), about 2% of replicated molecules undergo deletion of lambda-P(L) during transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of S(mu) increases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.