Three mutant immunoglobulin heavy chain (IgH) insertion mice were generated in which a targeted nonfunctional IgH passenger transgene was either devoid of promoter (pdelta) or was placed under the transcriptional control of either its own RNA polymerase II-dependent IgH promoter (pII) or a RNA polymerase I-dependent promoter (pI). While the transgene mutation-frequency (0.85%) in memory B cells of pI mice was reduced compared to that in pII mice (1.4%), the distribution and the base exchange pattern of point mutations were comparable. In pdelta mice, the mutation frequency was drastically reduced (0.09%). The mutation frequencies correlated with the levels of transgene-specific pre-mRNA expressed in germinal center B cells isolated from the mutant mice.

We have constructed the physical map of the 0.8 megabase DNA fragment which contains the 3' 64 variable region (V) gene segments of the human immunoglobulin heavy chain (H) locus. The organization of the VH locus showed several features that indicate dynamic reshuffling of this locus. The sequenced 64 VH segments include 31 pseudogenes, of which 24 are highly conserved except for a few point mutations. Comparison of the 64 germline VH sequences shows that each VH family has conserved sequences, suggesting that there might be some genetic or selection mechanisms involved in maintenance of each family. The total number of the human VH segments was estimated to be about 120, including at least 7 orphons.

How rearranged immunoglobulin (Ig) genes are further diversified by somatic hypermutation is unknown. Using VDJ passenger Ig heavy chain (IgH) knockin mouse strains, we now demonstrate a high frequency of DNA double-strand breaks (DSBs) in the targeted VDJ passenger gene of germinal center (GC) B cells. These DSBs parallel the distribution of mutations in the targeted hypermutation domain and are found preferentially at RGYW motifs, the intrinsic hot spots of somatic hypermutation. The introduction of DSBs appears to depend on transcriptional activity. Thus, secondary diversification of rearranged V gene segments relates to an error-prone nonhomologous DSB repair system acting in B cells of the GC.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vλ1 light chain genes from naive and memory B cells in DNA repair–deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand–break repair, and AAG-mediated base excision repair.

We have constructed the physical map of the 3′ region of the human immunoglobulin heavy chain variable region (VH) genes. DNA segments extending to 200 kb upstream of the JH segment were isolated in two YAC clones. Five VH segments were identified in this region in the 5′ to 3′ order, V(II‐5), V(IV‐4), V(I‐3), V(I‐2), and V(VI‐1) segments which were all structurally normal and orientated in the same direction as the JH segments. From DNA of a different cell line we have isolated a cosmid contig containing the same DNA region which has extraordinary polymorphism. The YAC and cosmid DNAs were called haplotypes A and B, respectively. Haplotype B contained an additional VH‐I segment (V(I‐4.1b)) between the V(II‐5) and V(IV‐4) segments. V(I‐4.1b) segment is almost identical to a previously published VH sequence encoding a rheumatoid factor. Another VH segment in the B haplotype (V(I‐3b)) corresponding to the V(I‐3) segment also showed 99.7% nucleotide sequence homology with an anti‐DNA autoantibody VH sequence. However, none of the VH sequences in haplotype A showed such strong homology with autoantibody VH sequences. The results suggest that VH haplotypes may have linkage with autoantibody production.