Site-directed cleavage of immunoglobulin gene segments by lymphoid cell extracts.
To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V-J joining in vitro. The DNA from a hybrid phage (lambda VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J-CK sequence was incubated with lymphoid cell extracts and packaged in vitro. Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance. Although no site-specific V-J fusion events were detected, the packaging efficiency of lambda VJCK DNA was 10(2)- to 10(3)-fold lower than that of lambda DNA. This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences. To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots. Double-stranded cleavages were observed to occur near the 5' end of each one of the five JK gene segments and near the 3' end of a VK gene segment. A plasmid containing the mouse I-A beta gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments.
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000502:physiology; D003090:Coliphages / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004622:Embryo, Mammalian; D004926:Escherichia coli; D007144:Immunoglobulin J-Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D008214:Lymphocytes / Q000276:immunology* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D011995:Recombination, Genetic
D002457:Cell Extracts; D004274:DNA, Recombinant; D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D004262:DNA Restriction Enzymes