NOVEL ELECTROPHORETIC PROTOCOL FOR COLLECTION OF MUTATIONS IN THE LAMBDA-LIGHT CHAIN IMMUNOGLOBULIN GENE IN A HUMAN B-LYMPHOBLASTOID CELL STRAIN

Spontaneous and chemically induced mutation was examined in the lambda light chain immunoglobulin gene in a human B-lymphoblastoid cell stra in (T5-1). The hemizygous lambda gene is a unique mutational target ge ne which codes for a protein that is both expressed on the cell membra ne and secreted. Mutations in the lambda gene were detected by analysi s of western blots of isoelectric focusing gel electrophoresis of T5-1 cell conditioned culture medium. None of 5,841 individual clones esta blished from vehicle-exposed populations had detectable variations in the isoelectric banding pattern of the constitutively secreted lambda immunoglobulin protein. In contrast, 113 of 6,128 clonal populations e stablished from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-exposed po pulations exhibited stable variations in expression of the lambda immu noglobulin: isoelectric variants (n = 3) and nonsecretors (n = 110). M NNG-induced mutations in the lambda gene, which resulted in lambda imm unoglobulin proteins with altered isoelectric points (pIs), occurred a t a frequency of no less than 4.9 x 10(-4) mutations/cell, indicating the mature rearranged lambda immunoglobulin gene is comparably sensiti ve to carcinogen induced mutation as other human autosomal target gene s. Approximately one-half of the MNNG-induced non-secretor mutant clon es lacked lambda mRNA while one-half maintained constitutive transcrip tion and expression of the lambda immunoglobulin an the cell surface, demonstrating that carcinogen damage interdicted gene function at mult iple points. (C) 1995 Wiley-Liss, Inc.