Fig. 4.

Genealogical trees generated from sequences of IgG1⁺ memory B cells transferred into three AID^−/− mice. Genealogical relatedness of the V_HD_HJ_H sequences of typical secondary memory B cells specified by clone numbers in spleen (blue circles) and BM (red circles) recovered from individual recipient (A, C and F from a; D, E and G from b and B and H from c) at 1 week after challenge are illustrated. RNA was purified from spleen and BM from individual mice, and rearranged V_H gene transcripts were amplified using V_H186.2-Cγ1 primer pairs and sequenced as described in Methods. Individual clones were assigned to a particular genealogical tree on the basis of having identical V_HD_HJ_H sequence and junctional diversity, together with shared and unique mutations. The numbers of mutated codons are indicated within circles. Clones carrying amino acid changes and those carrying silence mutations compared with their hypothetical parent clone are indicated by red and black characters, respectively. The length of each branch represents the number of mutations. Hypothetical clone members (black circles) are deduced from the sequences of internal clones. The clone with a dotted line represents the one carrying a V_H gene with a stop codon generated by somatic mutations. Purple circles represent clones identified in both spleen and BM. Circles with double and bold lines represent clones carrying an amino acid substitution from Trp to Leu at amino acid 33 of CDR1 and Lys to Arg at amino acid position 58 of CDR2, respectively. The clone name is shown below the genealogical tree and the sequence represents D_H and J_H regions that are underlined and in italics, respectively. The red sequence represents DFL16.1.