This theoretical estimate of combinatorial diversity is based on the number of germline V gene segments contributing to functional antibodies (see Fig. 5.4); the total number of V gene segments is larger, but the additional gene segments are pseudogenes and do not appear in expressed immunoglobulin molecules. In practice, combinatorial diversity is likely to be less than one might expect from the calculations above. One reason is that not all V gene segments are used at the same frequency; some are common in antibodies, while others are found only rarely. This bias for or against certain V gene segments relates to their proximity with intergenic control regions within the heavy-chain locus that activate V(D)J recombination in developing B cells. Also, not every heavy chain can pair with every light chain: certain combinations of VH and VL regions will not form a stable molecule. Cells in which heavy and light chains fail to pair may undergo further light-chain gene rearrangement until a suitable chain is produced or they will be eliminated. Nevertheless, it is thought that most heavy and light chains can pair with each other, and that this type of combinatorial diversity has a major role in forming an immunoglobulin repertoire with a wide range of specificities.
5-8 Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to the diversity of the third hypervariable region.
As noted earlier, of the three hypervariable loops in an immunoglobulin chain, CDR1 and CDR2 are encoded within the V gene segment. CDR3, however, falls at the joint between the V gene segment and the J gene segment, and in the heavy chain it is partly encoded by the D gene segment. In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments. The added nucleotides are known as P-nucleotides and N-nucleotides, and their addition is illustrated in Fig. 5.11.
P-nucleotides are so called because they make up palindromic sequences added to the ends of the gene segments. As described in Section 5-5, the RAG proteins generate DNA hairpins at the coding ends of the V, D, or J segments, after which Artemis catalyzes a single-stranded cleavage at a random point within the coding sequence but near where the hairpin was first formed. When this cleavage occurs at a different point from the initial break induced by the RAG1/2 complex, a single-stranded tail is formed from a few nucleotides of the coding sequence plus the complementary nucleotides from the other DNA