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 strain (T5-1). The hemizygous lambda gene is a unique mutational target gene which codes for a protein that is both expressed on the cell membrane and secreted. Mutations in the lambda gene were detected by analysis of western blots of isoelectric focusing gel electrophoresis of T5-1 cell conditioned culture medium. None of 5,841 individual clones established 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 established from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-exposed populations exhibited stable variations in expression of the lambda immunoglobulin: isoelectric variants (n = 3) and non-secretors (n = 110). MNNG-induced mutations in the lambda gene, which resulted in lambda immunoglobulin proteins with altered isoelectric points (pIs), occurred at a frequency of no less than 4.9 x 10(-4) mutations/cell, indicating the mature rearranged lambda immunoglobulin gene is comparably sensitive to carcinogen induced mutation as other human autosomal target genes. Approximately one-half of the MNNG-induced non-secretor mutant clones lacked lambda mRNA while one-half maintained constitutive transcription and expression of the lambda immunoglobulin on the cell surface, demonstrating that carcinogen damage interdicted gene function at multiple points.
McFarland
R D
RD
Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill 27599-7525, USA.
Smith
G J
GJ
D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D015153:Blotting, Western; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D016130:Immunophenotyping; D007525:Isoelectric Focusing; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D016296:Mutagenesis; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured
D007147:Immunoglobulin Light Chains; D012333:RNA, Messenger; D008769:Methylnitronitrosoguanidine