ANALYSIS OF INDIVIDUAL IMMUNOGLOBULIN-LAMBDA LIGHT-CHAIN GENES AMPLIFIED FROM SINGLE CELLS IS INCONSISTENT WITH VARIABLE REGION GENE CONVERSION IN GERMINAL-CENTER B-CELL SOMATIC MUTATION

Responding B cells in specific immune responses diversify their immuno globulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechan istic hypotheses of either error-prone DNA synthesis or templated vari able region gene conversion as the underlying mechanism in the generat ion of these mutations. To assess the role of gene conversion in germi nal-center somatic mutation, we chase to examine nucleotide changes in mouse lambda light chain genes which arose in response to a specific antigen. Laboratory mice possess three V lambda subexons, two of which differ from one another by only seven nucleotides, making these two s ubexons ideal for gene conversion. In the current study, we used six-p arameter flow cytometry to isolate single lambda light chain-expressin g germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individu al V(lambda)1 genes from these single cells for mutational analysis. N one of the 32 V(lambda)1 genes, containing a total of 40 mutations, sh owed evidence of gene conversion from either of the other V lambda sub exons. Features such as the replacement to silent ratio of the mutatio ns documented at the earlier time point indicate an absence of antigen -driven selection. These data indicate that V region gene conversion d oes not contribute to germinal-center somatic mutation and that gene c onversion is not responsible for targeting mutation specifically to re arranged Ig genes. The biological implications are discussed.