Volume 12, Issue 2, Pages 501-508 (August 2003) C-Terminal Deletion of AID Uncouples Class Switch Recombination from Somatic Hypermutation and Gene Conversion  Vasco Barreto, Bernardo Reina-San-Martin, Almudena R. Ramiro, Kevin M. McBride, Michel C. Nussenzweig  Molecular Cell  Volume 12, Issue 2, Pages 501-508 (August 2003) DOI: 10.1016/S1097-2765(03)00309-5

Figure 1 AIDΔ189-198 Induces Cytidine Deamination in DNA Kan resistance assay for AID-mediated cytidine deamination in UNG-deficient BH156 E. coli. Circles represent the mutation frequency of individual starting colonies; horizontal bars represent mean values. Results from two independent experiments are shown. Mean frequencies of Kanr colonies are indicated underneath each group. AID and AIDΔ189-198 were derived from pASK-AIDE. coli (Ramiro et al., 2003). Molecular Cell 2003 12, 501-508DOI: (10.1016/S1097-2765(03)00309-5)

Figure 2 Analysis of Gene Conversion and SHM in Retrovirally Infected AID−/− DT40 Cells (A) Map of the retrorival construct used to infect eukaryotic cells; GFP is translated from an IRES and its expression is correlated with AID expression; expression of the puromycin resistance gene allows for selection of infected cells. (B) Representative flow cytometry profiles of retrovirally infected AID−/− DT40 cells analyzed for surface IgM and GFP expression after 22 days of culture. (C) Proportion of IgM+ cells in infected (GFP+) AID−/− DT40 cells plotted against time in culture. Cells were infected, selected with puromycin, and plated (day 0) in triplicate (error bars refer to the standard deviation between triplicates). (D) Comparison of 40 randomly selected Vλ1 gene sequences cloned from AID−/− DT40 cells infected with AID or AIDΔ189-198encoding retroviruses and sorted for GFP expression after 24 days of culture. Each horizontal line represents the rearranged Vλ1/Jl (427 bp) with mutations classified as point mutations (lollipop shape), gene conversion tracts (horizontal bar above line), or single nucleotide substitutions, which could be a result of point mutation or gene conversion (ambiguous, vertical bar). Hollow boxes indicate deletions. (E) Proportion of Vλ1 sequences carrying different numbers of gene conversions (GC), point mutations (PM), or mutations of ambiguous origin (Amb) among GFP+ sorted populations. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of gene conversions or mutations indicated in the periphery of the charts. The total number of independent sequences analyzed is indicated in the center of each chart. Deletions, duplications, and insertions were excluded from this analysis. Molecular Cell 2003 12, 501-508DOI: (10.1016/S1097-2765(03)00309-5)

Figure 3 AIDΔ189-198 Fails to Induce CSR (A) Flow cytometry analysis of CSR in AID−/− mouse B cells stimulated with IL-4 + LPS and infected with AID-expressing retroviruses. CSR was measured by IgG cell surface expression and infected cells were GFP+. (B) Results from four independent experiments showing the percentage of IgG+ cells, among the GFP+ population. (C) Representative flow cytometry profiles of AID−/− B cells stimulated with IL-4 + LPS and infected with retroviruses encoding ER-AID fusion proteins, cultured in the absence or presence of tamoxifen (OHT). Numbers show mean value and standard deviation of the percentage of IgG+ cells within the GFP+ population from four cultures. Molecular Cell 2003 12, 501-508DOI: (10.1016/S1097-2765(03)00309-5)

Figure 4 Mutations in Sμ (A) Proportion of sequences carrying different number of mutations in Sμ. Sμ was cloned from retrovirally infected GFP+ AID−/− B cells that had been stimulated with LPS and IL-4 that were purified by cell sorting. The frequency of mutations per bp sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart, respectively. The number of mutations was: vector only, 1 mutation/34,160 bp; AID, 65 mutations/56,567 bp; and AIDΔ179-198, 62 mutations/58,209 bp. Statistical significance was determined by a two-tailed t test assuming unequal variance. P values are indicated below each pie chart. (B) Distribution of point mutations in Sμ. The region sequenced is indicated with the first base corresponding to position 4600 in the Sμ germline sequence (Genbank accession number J0040); allotypic differences were taken into consideration and not scored as mutations. Independent mutations are shown in lowercase letters above and below the line for AID and AIDΔ189-198, respectively. Hotspot motifs (Rogozin and Kolchanov, 1992) containing mutations are shown in bold. There were three deletions in each group. Sequences were pooled from two independent experiments. Molecular Cell 2003 12, 501-508DOI: (10.1016/S1097-2765(03)00309-5)