Modified AIDs give altered IgVλ hypermutation spectra in B cells. (A) Hypermutation of IgVλ was assayed by monitoring surface IgM-loss in AID^−/− ψV^−/− sIgM⁺ DT40 cells that had been stably transfected with constructs coexpressing the indicated AID mutants together with GFP. For each construct, the percentage of surface IgM-loss variants in 8–12 independent clonal transfectants were determined 3 wk after subculturing. On the right, Western blots representative of multiple clones show AID abundance in the DT40 cell extracts, with tubulin as loading control. (B) 5′-flanking nucleotide preferences of the IgVλ C mutations produced by the variant AID deaminases in the DT40 clonal transfectants. The compilations are based on mutations detected in unsorted DT40 cells analyzed 8 wk after transfection, except for AID/3C, where sequences from both unsorted and sorted sIgM⁻ populations contributed to the mutation database. In the case of AID¹*/3G, the nucleotide preferences are given based on an analysis of all the mutations in the dataset, as well as from an analysis in which the four major hotspots were removed from the calculations. (C) Percentage of mutated C residues flanked by 5′-purine (red) or 5′-pyrimidine (blue) in IgVλ sequences analyzed from individual expanded DT40 clonal transfectants represented by each bar. (D) Distribution of IgVλ mutations in the DT40 transfectants, in each comparing the spectrum achieved with a modified AID (below the line) to that achieved with wild-type AID (above the line). Mutations (which were >95% at C:G pairs) were computed as being caused by C deamination with those Cs flanked by a 5′-purine (Pu-C) indicated in red and those by a 5′-pyrimidine (Py-C) in blue. Further details on the mutations obtained with these deaminases, as well as with AID¹ are shown in Fig. S2 and Fig. S3.