Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.

Abstract

The rate and nature of hypermutation of immunoglobulin genes are of prime importance in the affinity maturation of antibodies. Although a considerable body of information has been gathered for K light chains, there is much less data for A chains. We have derived a large data base of somatic mutants of mouse Al light chains from Peyer's patches germinal center B cells. The endogenous Al genes mutate at a rate comparable to that previously found for a K transgene (V,coxl). There are intrinsic hot spots of mutation common to both in-frame and out-of-frame rearrangements; these hot spots cluster in hypermutating domains. In contrast to the pattern seen for VKOxl, the hot spot clusters are found not only in complementarity-determining region (CDR)l but also in CDR2 and CDR3; mutations also cluster in the joining/constant region intron. The differences between the pattern of mutations in VicOxl and Al light chains are discussed.Somatic hypermutation is a key element in antibody diversification and in the maturation of the immune response. Although first demonstrated in A chains (1), somatic mutation has not been as intensively studied as in the heavy-and K light-chain loci (2-4). Indeed, only a small data base of A mutations has been accumulated, and A genes have been suggested to mutate at lower rates than some heavy-chain variable region (V) and K light-chain V genes (2). The A locus is the smallest of the mouse immunoglobulin loci, and germline sequence differences together with junctional imprecision give relatively little diversity. We have recently shown that sequence analysis of V genes cloned from Peyer's patches germinal center B cells provides a rapid way to analyze somatic hypermutation (5). The simplicity of the A locus makes it an easy target for analysis with this approach, and we present the results of such a study.t MATERIALS AND METHODSIsolation of Peyer's Patches A+ B Cells. Peyer's patches were isolated from pooled CBA x C57BL/6 mice (6-12 mo old). The K+ cells were removed by incubation with biotinylated rat anti-mouse K (187.1) antibody (6) followed by streptavidin-magnetic beads (Dynal, Oslo). Germinal center B cells [CD45R(B220)+, peanut agglutinin (PNA)hi] were then sorted as described (5).DNA Isolation and Amplification. DNA was isolated from sorted cells (105), and the rearranged VA genes were amplified by PCR and cloned into M13 essentially as described for VKOX1 (5), except that the extension was for 3 min at 72°C and that oligonucleotides used were primer 1 (5'-CTCGAAT-TCATGGCCTGGATTTCACTTAT), which primes in the leader region and contains an EcoRI site, and primer 2 (5'-CTCGGATCCTTCAGAGGAAGGTGGAAACA), which primes in constant region (C)Al and contains a BamHI site (7). Individual clones were sequenced with LAMBCKF2 (5'-GTCCAAGAAAAACCAGATCATTTATTCAC) and LAMBDAJ1B (5'-GTGAATAAATGATCTGGTTTTTCT-TGGAC) to prime in the frameworks and LAMBDAJ1 (5'-CTCGGATCCACTCACCTAGGACAGTCAGT) and LAMINFOR (5'-GGAGCAGTCTGAAATGAGACAAAG-CAT) to prime in the joining region (J)/C intron. RESULTSBoth In-Fra...

Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation

“…This and the following A of Met introduced a TA sequence. This observation supports the view that TA is part of an alternative motif involved in the origin of hot spots (30,34).…”

“…Indeed, A hot spots are generally preceded by T (unpublished results), and it is not rare to find the second base of tyrosine (TAT͞C) highly mutated. TA hot spots are, of course, also associated with other residues and present in untranslated segments (30,34).…”

“…30), and also with secondary structure features of the DNA, like palindromic sequences, direct and inverted repeats, etc. (31)(32)(33)(34). In this paper we show that key silent changes in the nucleotide sequence of hot spots have highly significant effects on the frequency at which individual bases hypermutate.…”

Evolution and the molecular basis of somatic hypermutation of antigen receptor genes

“…However, G:C bias is not a characteristic of the nurse shark NAR (or in the N53 shark L chain; Lee et al 2000) in spite of the absence of germinal centres (Diaz et al 1999). In addition, the pattern of mutation in passenger Ig transgenes of mice and/or humans, the heavy chain intron, non-Ig sequences placed within an Ig construct, and non-productively rearranged Ig genes, do not display a G:C bias (Azuma et al 1993;Betz et al 1993a;Gonzalez-Fernandez et al 1994;Yelamos et al 1995;Dorner et al 1997). Interpreting the G:C bias generated in mice de¢cient in MMR has been confounded by pleiotropic e¡ects, including microsatellite instability, that may impair proliferation in the germinal centre reaction (Vora et al 1999).…”

Transcription-Dependent Somatic Hypermutation Occurs at Similar Levels on Functional and Nonfunctional Rearranged IgH Alleles

“…21). Regarding allelic exclusion, the relative transcription rate of Ig loci in wild-type B cells carrying two VDJ rearrangements has not been studied; by contrast, the presence of somatic mutations within out-of-frame rearranged V genes has been documented (22,23). Since functional and nonfunctional Ig sequences are strongly related and cannot be individually studied by the means of hybridization techniques, we thus decided to use the accumulation of mutations for the quantitative evaluation of the relative transcription rates of both alleles in primary B cells that carry double VDJ rearrangements.…”