The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B ceUs is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. The DNA sequences both within and flanking rearranged antibody variable-region genes (VLJL and VHDJH) expressed in B lymphocytes can mutate at a very high rate (14; for recent reviews, see reference 28). This type of genetic variation is termed somatic hypermutation to distinguish it from the other more familiar processes of immunoglobulin diversification, such as junctional diversity, combirnatorial recombination, and the association of fully assembled heavy and light polypeptide chains.The mechanism of hypermutation in mammalian B cells is unknown, but several models have been proposed. These can be segregated into those models in which the DNA is the direct substrate for mutation (5,8,14,17), including gene conversion (26), or those in which the DNA becomes hypermutable as a consequence of gene expression (i.e., transcription-based models [41]). In the first type of model, the DNA is mutated directly either via specific nicking and error-prone repair enzymes (8, 14), through errors introduced by misalignment of replicating DNA templates directed by inverted or direct repeat sequences (17), or via localized amplification of DNA replication which generates errors at the normal rate but in greater number in a localized region (5). Alternatively, the model invoking gene conversion envisages that sequence information donated by other related genes is incorporated by recombination into the rearranged V gene (26). However, these DNA-based models do not state how the enzymes display the specificity which limits the mutational process to the DNA within and around rearranged V genes (41). Known DNA polymerases also display a very high copying fidelity in vivo (24,31,32). Thus, the DNA-based models also require the presence of new enzymes with V-region specificity.A second type of model based on gene expression ac- (15,18,20,22,30,39). They are not found in constant region sequences (15) and are found rarely in unrearranged germ line V genes (44). It is important now to precisely define the distribution of somatic mutations in and around rearranged V genes, particularly in the 5' upstream region, because this will help to identify the target region of the DNA and therefore influence ideas on the type of mutational mechanism involved. In this work, we contribute to a better definition of this target region by comparing DNA sequences of related sets of hypermutated VH genes with their germ line counterparts. MATERIALS AND METHODSCloning and sequencing rearranged somatically mutated VH genes. Genes derived from the VH186.2 germ line gene 5187 on May 12, 2018 by guest