A HYPERMUTABLE INSERT IN AN IMMUNOGLOBULIN TRANSGENE CONTAINS HOTSPOTS OF SOMATIC MUTATION AND SEQUENCES PREDICTING HIGHLY STABLE STRUCTURES IN THE RNA TRANSCRIPT

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can underg o somatic hypermutation upon cell interaction with antigen and T cells . The mutation mechanism had previously been shown to depend upon tran scription initiation, suggesting that a mutator factor was loaded on a n RNA polymerase initiating at the promoter and causing mutations duri ng elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (E PS) located within the variable (V) region of an Ig transgene. This su bstrate can easily be assayed for the presence of mutations in DNA fro m transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites h ave been altered. Surprisingly, the EPS insert was mutated many times more frequently than the nanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutatio n where the fine specificity of the mutations is determined by nucleot ide sequence preferences of a mutator factor, and where the general si te of mutagenesis is determined by the pausing of the RNA polymerase d ue to secondary structures within the nascent RNA.