Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin...

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 PL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S mu-lambda PL-S gamma 3, about 2% of replicated molecules undergo deletion of lambda PL 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.