Distribution of Sμ-associated mutations. (A) Comparison of switch-associated mutations in wild-type and msh2^−/− ung^−/− mice. Mutations in Sμ-associated regions m.1 and m.2 and in Sα-associated region a.2 were compared with Peyer's patch germinal center B cells from msh2^−/− ung^−/− and wild-type mice. Mutations were categorized as either transitions at C:G pairs (plotted above the midline) or other (transversions at C:G or transitions/transversions at A:T, plotted below the midline). Mutations are plotted as vertical bars, with the height representing the frequency of mutations at each nucleotide calculated as the number of cases per set of 21 sequences in which that position is mutated. (B) Distribution of transition mutations at C:G pairs in germinal center B cells from Peyer's patches of a pair of msh2^−/− ung^−/− mice (I and II). Four regions of ∼1.5 kb (m.1 to m.4) spanning from immediately downstream of J_H4 to within the C_μ4 exon were sequenced (Table I). Mutations are plotted as vertical bars, with the height representing the frequency of mutations at each nucleotide calculated as the number of cases per set of 11 sequences in which that position is mutated. Position 1 in the sequence represents nucleotide 134,071 (available from GenBank/EMBL/DDBJ under accession no. AC073553). (C) Distribution of mutations in the vicinity of the Eμ enhancer. The data are represented as a composite of the transition mutations at C:G pairs identified in mice I and II, though similar distributions are evident when each mouse is analyzed individually. The region encompassing the heterogeneous start sites of the μ germline transcripts (GLT) is indicated. The asterisks designate the extents of the segments highlighted.

Absence of major strand polarity in Sμ-associated mutations. (A) Transition mutations at C:G pairs in regions m.1to m.4 were categorized as mutations at C on either the transcription template or nontemplate strand (TS or NTS, respectively) and accordingly plotted above or below a midline. (B) An expanded 720-bp region depicts in detail the distribution of NTS versus TS deaminations identified in four clones amplified by nested PCR from the repetitive region of Sμ. Position 1 corresponds to nucleotide 137,456 (available from GenBank/EMBL/DDBJ under accession no. AC073553). Of the 148 C→T transition mutations detected, 130 fell within a WRC motif, 49 were on the nontranscribed strand, and 99 were on the transcribed strand. Of the WRC motifs in this 720-bp region, 55% are found on the transcribed strand.

DNA strand polarity of switch-associated mutations in LPS-cultured B cells. (A) DNA strand polarity of C→T transition mutations in individual S region sequences amplified from splenic B cells from msh2^−/− ung^−/− mice at day 7 of culture with LPS+IL-4/IFN-γ. (B) DNA strand polarity of C→T transition mutations in individual Sμ m.2 sequences amplified from splenic B cells from msh2^−/− ung^−/− mice at day 5 of culture with LPS+IL-4/IFN-γ. All mutated sequences are illustrated with C→T transitions on the transcription template strand indicated by open circles below the line; those on the nontemplate strand are indicated by closed circles above the line. Asterisks mark sequences that harbor tandem C→T transitions on one strand in regions where other sequences in the database exhibit mutations on the opposite strand. Clusters of same DNA strand (linked) mutations were quantified by scoring the frequency of “same” (AA to TT) contiguous mutations (ignoring intervening distance) compared with the frequency of “different” (AT or TA) contiguous mutations in each sequence obtained from LPS-stimulated B cells. The bias in favor of same strand mutations was estimated using a χ² test (P = 0.0277).

Distribution of Sγ-, ɛ-, and α-associated mutations. The distribution of transition mutations at C:G pairs around Sγ, ɛ, and α in the Peyer's patch germinal center B cells of msh2^−/− ung^−/− mice I and II are presented as a compilation. Mice I and II showed similar distributions of mutations across the IgH locus when analyzed separately and contribute 143 and 210 sequences, respectively, to the compilation. The regions analyzed (0.8–1.9 kb in length) are indicated by opposing arrowheads. The results for each isotype are aligned to the major start sites of the germline transcripts, which are taken as nucleotides 41,758 (γ3), 68,759 (γ1), 93,343 (γ2b), and 111,899 (γ2a; available from GenBank/EMBL/DDBJ under accession no. AC161365); nucleotide 6225 (ɛ; available from GenBank/EMBL/DDBJ under accession no. CAAA01071548); and nucleotide 126 (α; available from GenBank/EMBL/DDBJ under accession no. D11468). Mutations are plotted as vertical bars with the height representing the frequency of mutations at each nucleotide calculated as the number of cases per set of 22 sequences in which that position is mutated.

Cytokine induction of switch-associated mutations in LPS-cultured B cells. (A) In vitro class switching of splenic B cells from a wild-type (control) C57BL/6 mouse analyzed by flow cytometry after a 7-d culture with LPS plus either IL-4 or IFN-γ. The numbers indicate the percentage of events in the top right quadrants. Results are representative of two independent experiments. (B) Germline transcription in the in vitro–stimulated splenic B cells. Germline transcripts comprising I_H and C_H exons of each isotype were detected by RT-PCR from RNA extracted at day 3 of culture. hprt, hypoxanthine phosphoribosyltransferase control. (C) Distribution of transitions at C:G pairs in genomic DNA purified from splenic B cells from msh2^−/− ung^−/− mice after a 7-d culture with LPS plus IL-4 or IFN-γ as indicated. For each isotype, a region that had been demonstrated to be targeted by AID in vivo (Fig. 2) was amplified and sequenced. The number of sequenced for each region vary as a consequence of sequencing failures. Data shown are from two independent experiments.