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.

We present here a unifying hypothesis for the molecular mechanism of somatic hypermutation and somatic gene conversion in IgV genes involving reverse transcription using RNA templates from the V-gene loci to produce cDNA which undergoes homologous recombination with chromosomal V(D)J DNA. Experimental evidence produced over the last 20 years is essentially consistent with this hypothesis. We also review evidence suggesting that somatically generated IgV sequences from B lymphocytes have been fed back to germline DNA over evolutionary time.

We have used the polymerase chain reaction (PCR) in an attempt to clone and sequence the exons and hitherto unavailable contiguous flanks of all members of the small V(H) 9 germline gene family from inbred mouse strains and sublines that have had a common ancestry within the last century, and to analyze the molecular evolution of these sequences. Fifteen genuine germline genes were isolated (designated V(H) 9.1 through V(H) 9.15) from strains and sublines of DBA, BALB, 129 and C57BL inbred mice. Of the 15 genuine isolates, nine are novel: seven sequences from DBA strains and sublines ( V(H) 9.3 to V(H) 9.9) and two sequences from C57BL strains ( V(H) 9.13 and V(H) 9.14). We have identified sequencing errors and PCR recombinant artefacts in previously published sequences. We detected no sequence divergence of individual genes shared by the strains and sublines studied. However, we isolated two genes from DBA strains and sublines, V(H) 9.1 and V(H) 9.3, that differ only by five nucleotides encoding three amino acid changes that are concentrated within a 33 nucleotide (11 codon) region. Of these 11 codons, eight encode a putative antigen binding site. There were no differences in the remaining 733 nucleotides sequenced (including both 5' and 3' flanking regions). Potential explanations for the generation of V(H) 9.1 and V(H) 9.3 are discussed.

Summary In human and mouse, the germline contains a tandem array of highly homologous variable (V) gene elements which encode part of the antigen-binding region of the antibody protein. During evolution this array apparently arose by gene duplication followed by diversi®cation of duplicated genes via point mutation and recombination. Analysis of germline V gene sequences using a novel algorithm shows that major recombination sites coincide with the borders of the leader intron and the cap site, consistent with the hypothesis that over evolutionary time cDNA derived by reverse transcription of pre-mRNA in B lymphocytes has recombined with germline DNA.