V(H) replacement in rearranged immunoglobulin genes

Abstract

Examples suggesting that all or part of the V(H) segment of a rearranged V(H)DJ(H) may be replaced by all or part of another V(H) have been appearing since the 1980s. Evidence has been presented of two rather different types of replacement. One of these has gained acceptance and has now been clearly demonstrated to occur. The other, proposed more recently, has not yet gained general acceptance because the same effect can be produced by polymerase chain reaction artefact. We review both types of replacement including a critical examination of evidence for the latter. The first type involves RAG proteins and recombination signal sequences (RSS) and occurs in immature B cells. The second was also thought to be brought about by RAG proteins and RSS. However, it has been reported in hypermutating cells which are not thought to express RAG proteins but in which activation-induced cytidine deaminase (AID) has recently been shown to initiate homologous recombination. Re-examination of the published sequences reveals AID target sites in V(H)-V(H) junction regions and examples that resemble gene conversion.

Figure 1

Type 1 V_H replacement. (a) Usual subtype: recombination occurs after cleavage at the conventional RSS of a new (upstream) V_H and at the 3′ cryptic RSS in the V_H portion of the recombined V_H–D–J_H. (b)Cleavage occurs at the conventional RSS of the incoming V_H but at an upstream cryptic RSS of the V_H of the VDJ. (c) Cleavage occurs at cryptic RSS in opposite directions and different positions in the two V_H genes. In this and all other figures, the conventional RSS are indicated by filled triangles and cryptic RSS by open triangles. Small arrows indicate cleavage positions and deletion products are omitted. ‘∼’ indicates the typical random small deletions from either or both coding ends and added P and N bases.

Figure 2

Comparison of Types 1 and 2 V_H replacement. (a) Detail of the usual form of Type 1 involving the conventional RSS of the incoming V_H and the 3′ cryptic RSS in the V_H portion of the pre-existing V_H–D–J_H. Diagram conventions are as in Fig. 1. Just as in normal V(D)J recombination, the new junction in the VDJ is a coding-to-coding junction and the deletion product has a signal-to-signal junction. (b) A Type 2 V_H replacement scheme to explain the published hybrid sequences by the investigators' hypothesis of RAG-mediated double-strand cleavage at cryptic RSS and rejoining without end modification. Reaction products shown are a V_HDJ_H with the hybrid V_H of the kind published and the deduced circular deletion product, both with cryptic RSS signal-to-coding junctions. Alternative mechanisms are homologous recombination following RAG-mediated single-strand nicking at a cryptic RSS and our preferred hypothesis of AID-initiated homologous recombination unrelated to RAG or cryptic RSS. Homologous repair in these cases need not involve any deletion.

Figure 3

Construction of a sequence resembling Type 2 V_H replacement by PCR artefact. The N and P bases between V_H and D and between D and J_H are omitted for simplicity. (a) Copies of cDNA of two rearranged immunoglobulin heavy-chain genes occurring at an early stage in the same reaction mixture. ‘p’ is a PCR product made with a specific family V_H leader primer and a specific constant-region primer. ‘q’ is another rearrangement with a different D and J_H. The V_H differs from that of p at various points indicated by the pink spots in p and blue spots in q. q may be another PCR product with the same leader sequence, or it may be a cDNA template with a different leader that would not have been amplified with the primers in use. (b) During one of the PCR cycles, ‘n’, a copy of p, is incomplete when melting occurs. (c) In the next cycle n anneals with q at a point at which q has the same sequence as p. A polymerase molecule attaches and copying continues, giving a product (the extended ‘n’) that has the leader and 5′ end of the V_H the same as in p but the 3′ end of the V_H and the VDJ junction the same as q. This new molecule will now be amplified in all following cycles.