Recombination signal sequences (RSSs) are composed of heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by either 12 bp or approximately 23 bp of nucleotides. The heptamer–12-bp spacer–nonamer motif is depicted here as an orange arrowhead; the motif that includes the 23-bp spacer is depicted as a purple arrowhead. Joining of gene segments almost always involves a 12-bp and a 23-bp RSS—the 12/23 rule. The arrangement of RSSs in the V (red), D (green), and J (yellow) gene segments of heavy (H) and light (λ and κ) chains of immunoglobulin is shown here. The RAG-1 recombinase (see Section 5-5) cuts the DNA precisely between the last nucleotide of the V gene segment and the first C of the heptamer; or between the last G of the heptamer and the first nucleotide of the D or J gene segment. Note that according to the 12/23 rule, the arrangement of RSSs in the immunoglobulin heavy-chain gene segments precludes direct V-to-J joining.
family. Both the heavy-chain and the κ-chain V gene segments can be subdivided into seven families, and there are eight families of Vλ gene segments. The families can be grouped into clans, made up of families that are more similar to each other than to families in other clans. Human VH gene segments fall into three clans. All the VH gene segments identified from amphibians, reptiles, and mammals also fall into the same three clans, suggesting that these clans existed in a common ancestor of these modern animal groups. Thus, the V gene segments that we see today have arisen by a series of gene duplications and diversification through evolutionary time.
5-4 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences.
For a complete immunoglobulin or T-cell receptor chain to be expressed, DNA rearrangements must take place at the correct locations relative to the V, D, or J gene segment coding regions. In addition, these DNA rearrangements must be regulated such that a V gene segment is joined to a D or a J and not joined to another V gene segment. DNA rearrangements are guided by conserved noncoding DNA sequences, called recombination signal sequences (RSSs), that are found adjacent to the points at which recombination takes place. The structure and arrangements of the RSSs are shown in Fig. 5.6 for the λ and κ light-chain loci and the heavy-chain loci. An RSS consists of a conserved block of seven nucleotides—the heptamer 5ʹCACAGTG3ʹ, which is always contiguous with the coding sequence; followed by a nonconserved region known as the spacer, which is either 12 or 23 base pairs (bp) long; followed by a second conserved block of nine nucleotides, the nonamer 5ʹACAAAAACC3ʹ.
The sequences given here are the consensus sequences, but they can vary substantially from one gene segment to another, even in the same individual, as there is some flexibility in the recognition of these sequences by the enzymes that carry out the recombination. The spacers vary in sequence, but their conserved lengths correspond to one turn (12 bp) or two turns (23 bp) of the DNA double helix. This is thought to bring the heptamer and nonamer sequences to the same side of the DNA helix to allow interactions with proteins that catalyze recombination, but this concept still lacks structural proof. The heptamer– spacer–nonamer sequence motif—the RSS—is always found directly adjacent to the coding sequence of V, D, or J gene segments. Recombination normally occurs between gene segments located on the same chromosome. A gene segment flanked by an RSS with a 12-bp spacer typically can be joined only to one flanked by a 23-bp spacer RSS. This is known as the 12/23 rule.