title: High-frequency deletional rearrangement of immunoglobulin kappa gene segments introduced into a pre-B-cell line.
abstract: We describe an immunoglobulin gene recombination indicator in which a specific rearrangement via deletion results in the acquisition of a dominant phenotype. The indicator consists of the Escherichia coli xanthine/guanine phosphoribosyltransferase (gpt) gene, whose translation is prevented by the presence of an upstream initiation codon out of frame with respect to the gpt coding sequence. Flanking this barrier initiation codon are the heptamer-spacer-nonamer recognition sequences from a kappa chain variable region (V kappa) and from a kappa chain joining region (J kappa). A proper V-J joint results in the deletion of the translational barrier and allows expression of the selectable marker. When tested by transfection into fibroblasts, no rearrangements were detected and the presence of the barrier initiation codon was sufficient to completely abolish gpt expression in these cells. Similarly, no rearrangements were detected after transfer of the test gene into myeloma cells. However, when the construct was introduced into the pre-B-cell line 38B9, greater than 80% of the transfected cells showed evidence of a specific rearrangement. These rearrangements were associated with the translation of gpt, although no selection for its expression was needed. DNA sequence analysis of six different V-J joints revealed that the rearrangement proceeded with a high degree of accuracy. These results indicate that only very minimal DNA sequences (21 base pairs 5' of the V heptamer and 4 base pairs 3' of its nonamer; less than 45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that kappa gene segments can rearrange by a deletional mechanism.
mesh_terms: D000916:Antibody Diversity; D001402:B-Lymphocytes; D002460:Cell Line; D002872:Chromosome Deletion; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000032:analysis; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase; D007144:Immunoglobulin J-Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D007328:Insulin; D009101:Multiple Myeloma / Q000378:metabolism; D010430:Pentosyltransferases / Q000096:biosynthesis; D011384:Proinsulin / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013728:Terminator Regions, Genetic
chemical_list: D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007328:Insulin; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C013737:preproinsulin; D011384:Proinsulin; D010430:Pentosyltransferases; D007041:Hypoxanthine Phosphoribosyltransferase