Somatic Hypermutation and Junctional Diversification at Ig Heavy Chain Loci in the Nurse Shark

Abstract

We estimate there are ∼15 IgM H chain loci in the nurse shark genome and have characterized one locus. It consists of one V, two D, and one J germline gene segments, and the constant (C) region can be distinguished from all of the others by a unique combination of restriction endonuclease sites in Cμ2. On the basis of these Cμ2 markers, 22 cDNA clones were selected from an epigonal organ cDNA library from the same individual; their C region sequences proved to be the same up to the polyadenylation site. With the identification of the corresponding germline gene segments, CDR3 from shark H chain rearrangements could be analyzed precisely, for the first time. Considerable diversity was generated by trimming and N addition at the three junctions and by varied recombination patterns of the two D gene segments. The cDNA sequences originated from independent rearrangements events, and most carried both single and contiguous substitutions. The 53 point mutations occurred with a bias for transition changes (53%), whereas the 78 tandem substitutions, mostly 2–4 bp long, do not (36%). The nature of the substitution patterns is the same as for mutants from six loci of two nurse shark L chain isotypes, showing that somatic hypermutation events are very similar at both H and L chain genes in this early vertebrate. The cis-regulatory elements targeting somatic hypermutation must have already existed in the ancestral Ig gene, before H and L chain divergence.

The Evolution of Multiple Isotypic IgM Heavy Chain Genes in the Shark

“…1. Intradomain primers detecting all C1 (gCH1-5Ј/gCH1-3Ј) and C2 (CH2-5Ј/CH2-3Ј) exons were described previously (11). The combination GR5Ј (20% GR1: 5Ј-GTTTCTCTACCTCAGCAAT-3Ј and 80% GR2-5: 5Ј-GTTAGTCTMCCTCTGGAAT-3Ј) and JH5 (5Ј-TCACIGTCACCAT GGT-3Ј) detects all genomic V H from Groups 1-5.…”

“…The clones with G2A V H carried a C2 containing BstEII site, which distinguishes the C region of G2A from G2B (11). Some V H and C fragments were sequenced for confirmation.…”

“…The epigonal organ cDNA library of shark-33 was described in a previous publication (11). The phages were screened with V H and C H probes, and Ig sequence from selected lysates were subjected to amplification using T3 primer and a universal oligo targeting C H 2.…”

“…Our model is the nurse shark, which we estimate to have 15 IgM clusters (11), and in this study we have characterized them in two unrelated individuals, in genomic and cDNA libraries from shark-33 and a bacterial artificial chromosome (BAC) 3 library from the genetically unrelated shark-Y. We found there are 9 -12 functional genes, and the gene number can differ slightly between individuals.…”

Diverse Immunoglobulin Light Chain Organizations in Fish Retain Potential to Revise B Cell Receptor Specificities

“…The various reported teleost L chain clusters are tightly linked, and the distance between the C exon of one cluster and the downstream inverted V of the next cluster is Ͻ5 kb in trout L1, Ͻ5 kb in cod L1, Ͻ6 kb in catfish G, and Ͻ10 kb in catfish F. Although it is usually remarked in reviews that rearrangement events are limited to within a cluster, this really only applies to observations at elasmobranch Ig clusters, which are not close together. Intracluster IgM H chain recombination was demonstrated in three horn shark rearrangements of the 1113 IgH locus (42) and 16 nurse shark rearrangements of the G2-V1 and G2-V2 IgH loci (43). Further characterization of 56 nurse shark IgM H chain cDNA revealed that all were rearrangements of V gene segments from groups 1, 2, 4, and 5 (44) to their respective group-specific J gene segments (V. Lee and E. Hsu, unpublished results).…”