The existence of the N-region of immunoglobulins (Igs) was postulated when it was observed that nucleotide sequences of small, but variable, lengths identified at the VD and DJ junctions of Ig heavy chains had no counterpart in any of the V gene, D gene, or J gene segments in the germline. It turned out that these nucleotides were added at both the VD and the DJ junctions by the enzyme terminal deoxynucleotidyl transferase (TdT), which is specifically expressed in lymphocytes.
Direct evidence that N diversity was indeed the result of TdT activity was first obtained by transfection of a non-Ig-producing fibroblast cell line with a DNA vector containing an insert of unrearranged D and J segments. Gene rearrangement was observed, provided that a co-transfection with the RAG1/RAG2 recombinase activating genes was made. In the absence of TdT, no additional nucleotides were inserted. When co-transfection also included the TdT gene, N diversity was observed in the rearranged substrate. The ultimate proof came from gene targeting experiments, because mice rendered TdT-/-had heavy chains that no longer contained N diversity.
The results of these experiments are also in good agreement with analysis of B-cell differentiation that takes place in fetal liver and in bone marrow. In mouse fetal liver, TdT is not expressed and, as a result, no N diversity is seen in B cells that have differentiated in this organ. By contrast, TdT expression takes place in bone marrow, but only at precise steps of B-cell differentiation—that is, at the proB stage. This is the time when DJ rearrangements first take place, which correlates nicely with the major N diversity that accumulates at the D-J joint. It is still active whenever the V to DJ recombination takes place, but is then down-regulated. As a result, no or very limited N diversity is seen in the light chains. Recent data confirmed the presence of N diversity in some light chains and suggest that this occurs later in the periphery, whenever both the TdT gene and the RAG system are reactivated transiently, especially in the course of an antigenic stimulation. This is another example of the extraordinary plasticity of the mechanisms that ensure Ig diversity.
