DNA mutagenesis is generally considered harmful. Yet activated B cells normally mutate the Ig loci. Because this somatic hypermutation is potentially dangerous, it has been hypothesized that mutations do not occur throughout the genome but instead are actively targeted to the Ig loci. Here we challenge this longstanding and widely accepted hypothesis. We demonstrate that hypermutation requires no Ig gene sequences. Instead, activationinduced cytidine deaminase and other trans-acting hypermutation factors may function as general mutators. U pon antigenic stimulation of B lymphocytes, the variable (V) regions of Ig loci mutate at a rate orders of magnitude higher than the normal spontaneous rate. This somatic hypermutation requires activation-induced cytidine deaminase (AID) (1), an enzyme expressed exclusively in activated B cells of the germinal center (2). The enzyme converts deoxycytidine to deoxyuridine (3, 4). Except for a short motif, WRC (W ϭ A or T, R ϭ A or G) on the nontemplate DNA strand, purified AID has no sequence preference in vitro (4). In line with biochemical experiments, transgenic expression of AID in fibroblasts (5), hybridomas (6), and even bacteria (7, 8) causes hypermutation.It has been reasoned that hypermutation must be targeted to the Ig loci, because if mutations occurred at locations outside the V region, this would be dangerous and eventually lead to tumorigenesis. Thus Ig hypermutation has been regarded as the prime example of site-directed mutagenesis (9-13). Surprisingly, the V region itself is not needed for hypermutation (14). Instead, nearby enhancers and other sequences from the Ig intron have been proposed to direct hypermutation to the V regions (15)(16)(17)(18)(19).Recently, three genes that do not encode the V region, BCL-6 (20), B29, and mb1 (21), were reported to hypermutate in normal B lymphocytes. To explain these results within the framework of site-directed mutagenesis, it was proposed that these genes have unidentified ''Ig-like'' (20) or common cis-acting regulatory sequences (21). Furthermore, the T cell receptor in AIDtransgenic mice (22) and a GFP plasmid reporter in AIDtransgenic fibroblasts (5) were shown to hypermutate. Although these observations seem to conflict with the notion of sitedirected mutagenesis, it has been suggested that transgenic overexpression of AID or lack of B cell factors leads to nonphysiological, nontargeted hypermutation (13).The paradigm of site-directed mutagenesis is deeply rooted. Despite some observations to the contrary, the interpretation of results has rarely sought to dispute this paradigm. Yet the putative factors that recruit and deliver AID to the V regions remain undiscovered. Given the nondiscriminating enzymatic activity of AID, we hypothesized that hypermutation is not an actively targeted process. Here we challenge the paradigm of site-directed mutagenesis by demonstrating that AID-mediated hypermutation requires no Ig gene elements. Materials and MethodsReporter Constructs. Constructs were based on the Moloney murine ...

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent ⌬RBCS1B::LUC chimeric gene fusion, lacking all 5 transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc ؉ ) required a homologous recombination event between the ⌬RBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B͞ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5 transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc ؉ seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc ؉ seedlings were isolated, and molecular and genetic analysis indicated that the luc ؉ trait was caused by the formation of a recombinant chimeric RBCS3B͞1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuff ling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossingover (Ϸ3 ؋ 10 ؊6 ), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.Genome organization can directly affect the evolution of a gene. Single-copy genes or dispersed members of a multigene family evolve independently. In contrast, members of a multigene family organized as a gene cluster can exploit this organization to generate further gene duplications and novel recombinant genes by a process of unequal crossing-over. For example, a single intergenic unequal crossover event in a gene cluster results in four simultaneous alterations: a deletion, a duplication, and two reciprocal, recombinant genes. The impact of such unequal crossover events evidently have been important in the evolution of complex loci such as HOX (1), amylase (2), globin (3), MHC (4), Ig (5), the maize R-r complex (6), RBCS (7), and plant disease-resistance loci (8-10). Although DNA sequencing of gene clusters provides information about past changes in a particular gene cluster, it can only estimate the rate of unequal crossing-over in terms of geological time scales.In multicellular organisms, unequal crossing-over is implicated in several genetic disorders. This was demonstrated first with the Drosophila bar locus (11). Subsequent research with the bobbed locus demonstrated ...