No abstract

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

Summary Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be ampli®ed simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between dierent germline sequences during ampli®cation are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of ampli®cation cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of ampli®cation cycles, becoming a high proportion of the total number of PCR products once the`plateau phase' of the reaction was reached. Recombination events were located throughout the $ 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of ampli®cation cycles), recombination artefacts can be virtually eliminated from PCR ampli®cations involving mixtures of very similar sequences. This information is relevant to all studies involving PCR ampli®cation of members of highly homologous multigene families of cellular or viral origin.

SummaryWe have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase-η exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol-η and two other Y-family enzymes that are dispensable for SHM, human pols-ι and -κ , copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol-η was confirmed using a second sample from an independent source. Human DNA pols-β and -µ , and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-η was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro . It is thus feasible that pol-η acts as both a RNA-and a DNA-dependent DNA polymerase in SHM in vivo , and that Y-family RT activity participates in other mechanisms of physiological importance.