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 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.
Leung
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Department of Molecular Biophysics and Biochemistry, Yale Medical School, New Haven, CT 06510.
Maizels
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D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D005812:Genes, Switch; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic