Cell-free recombination of immunoglobulin switch-region DNA with nuclear extracts.
We have developed an in vitro recombination system employing cell-free nuclear extracts from human B lymphocytes capable of detecting the recombination between human mu switch (Smu) and Sepsilon sequences in a model plasmid. Nuclear extracts from CD40-stimulated B lymphocytes gave a higher frequency of recombination in the assay than the unstimulated B cells. Recombination between Smu and Sepsilon was mediated by the nuclear extracts as the recombinational products could be amplified prior to bacterial transformation. Characterization of the recombination products demonstrated that the recombination process had the characteristics of immunoglobulin (Ig) isotype switching, as it was (i) switch-region-sequence specific, (ii) nonhomologous recombination, and (iii) enhanced by CD40 stimulation. Transcription through the S region DNA was not required for recombination in the system. These results demonstrate that Ig switch-region DNA recombination can be accomplished in vitro by cell-free nuclear extracts. This in vitro system for Ig switch-region DNA recombination using cell-free nuclear extracts will permit the dissection of the events involved in IgE class switch recombination, a critical event in the development of allergic diseases.
Zhang
K
K
The Hart and Louise Lyon Laboratory, UCLA School of Medicine, Los Angeles, California 90095-1680, USA. kzhang@mednet.ucla.edu
Cheah
H K
HK
D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D019013:CD40 Antigens / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D017578:Immunoglobulin Class Switching; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000145:classification; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000276:immunology; D020080:Tandem Repeat Sequences / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D019013:CD40 Antigens; D007074:Immunoglobulin G