Antibody diversity: Somatic hypermutation of rearranged V_H genes
The immune response to phosphorylcholine in BALB/c mice has been well characterized. Amino acid sequence analyses of heavy-chain variable (V_H) regions from 19 myeloma and hybridoma immunoglobulins binding phosphorylcholine show that 10 are identical (the prototype T15 V_H sequence) and 9 are distinct variants differing by one to eight residues. A T15 V_H DNA probe was used to isolate four closely related members of the T15 V_H gene family, including one encoding the T15 V_H sequence, from a sperm genomic library. A comparison of the protein and germline V_H sequences suggested that most of the immune response to phosphorylcholine is derived from the T15 germline V_H gene segment. The variant heavy chains from the M167 and M603 α immunoglobulins differ in their V_H protein sequences from T15 by eight and three residues, respectively. We analyzed the somatic variability in and around the coding regions of these two variant V_H genes by comparing them with the corresponding regions of the appropriate germline gene segments. The somatic variation has three properties: it is extensive and is found in flanking as well as coding sequences (for example, at least 44 substitutions for the M167 sequence and 10 substitutions for the M603 sequence); in the coding regions, it includes many silent as well as replacement substitutions; and it is focal in nature and centered around the rearranged V_H genes. Although the mutations extend into the neighboring upstream and downstream flanking sequences, sequences approximately 5 kb upstream and downstream from the V_H genes show no substitutions. Moreover, the associated heavy-chain constant genes (C_α from both variant α genes are unaltered, indicating that a closely linked and coexpressed gene is unmutated. We conclude that this somatic variation is generated by a special hypermutational mechanism highly localized in its site of execution and highly restricted in its time of operation during B-cell development.