STRUCTURE AND DIVERSITY OF THE HEAVY-CHAIN VDJ JUNCTIONS IN THE DEVELOPING MEXICAN AXOLOTL

The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2. 5-, 3.5-, 10-, and 24-month-old animals were sequenced, Six JH segment s were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Iph chains i n the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolot ls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid lo nger in 2-year-old adult animals (mean: 9.5 residues). Several pairs o f identical VDJ/CDR3 sequences were shared between 3.5-month-old indiv idually analyzed axolotls, or between groups of axolotl of different a ges. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the pr obability that different 3.5-month-old axolotl larvae would produce id entical junctions seems very low, considering their limited number of B cells (less than 10(5)). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens.