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 μ and γ3 chain switch (S)-region sequences, Sμand Sγ 3, are transiently introduced into primary murin...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1992-05, Vol.89 (9), p.4154-4158
Main Authors: Leung, Helios, Maizels, Nancy
Format: Article
Language:English
Subjects:
Animals
B lymphocytes
B-Lymphocytes - physiology
Biochemistry
Biological and medical sciences
Cells, Cultured
Cultured cells
Cytomegalovirus - genetics
Deoxyribonucleic acid
DNA
Enhancer Elements, Genetic
Fundamental and applied biological sciences. Psychology
Genes, Immunoglobulin
Genes, Switch
Genetics
Genic rearrangement. Recombination. Transposable element
In Vitro Techniques
Isotypes
Mice
Mice, Inbred BALB C
Molecular and cellular biology
Molecular genetics
Molecules
Plasmids
Promoter regions
Promoter Regions, Genetic
Recombination, Genetic
Regulatory Sequences, Nucleic Acid
Replication origin
Spleen cells
Transcription, Genetic
Transfection
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Summary: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 μ and γ3 chain switch (S)-region sequences, Sμand Sγ 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 λ (λPL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying Sμ-λPL-Sγ 3, about 2% of replicated molecules undergo deletion of λPLduring transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of Sμincreases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.89.9.4154