Hypermutation is observed only in antibody H chain V region transgenes that have recombined with endogenous immunoglobulin H DNA: implications for the location of cis-acting elements required for somatic mutation.

Abstract

SummaryMice with transgenes containing an antibody H chain V region (V.DJ.) gene were used in an analysis of the c/s-acting elements required for hypermutation of immunoglobulin (Ig) V genes. These transgenes can somatically recombine with endogenous IgH DNA, leading to the formation of functional heavy (H) chains partially encoded by the transgenic VsDJ.. The transgenomes in the five different lines of mice analyzed contain as little as 150 bp, and as much as 2.8 kb of natural DNA flanking the 5' side of the V. and either 1.5 or 2.3 kb (including the intronic enhancer and 5' matrix attachment region [MAR]) flanking the 3' side of Vs. Hybridomas were constructed from immunized transgenic mice, and transgenes present in these hybridomas that had or had not recombined to form functional H chain loci were sequenced. The data obtained show that: (a) the recombined transgenes contain hypermutated V. genes; and (b) among such transgenes, even those containing only 150 bp of natural V. 5' flanking sequence and several kilobases of 5' plasmid vector sequence display a frequency, distribution, and type of mutation characteristic of conventional IgH loci. The data also indicate that transgenic VsDJ. genes that have not recombined with endogenous IgH DNA are not substrates for hypermutation, even if they are flanked by 2.8 kb of natural 5' DNA, and 2.3 kb of natural 3' DNA, including the J.2-JH4 region, a MAR, and the intronic enhancer. Collectively, the data suggest that sequences 5' of the V. promoter are dispensable, a V. promoter and the intronic IgH enhancer region are not sufficient, and a region(s) within or 3' of the IgH constant region locus is requisite, for hypermutafion of Ig V. transgenes. ntibody V region diversity in mice and humans is generated by the combinatorial joining of germline V gene segments that are members of heterogeneous multigene families, the deletion and de novo addition of nucleotides at the junctions of these V segments during joining, and hypermutation of the resulting V genes (1). While characterization of the cis-and trans-acting factors involved in the rearrangement of V gene segments and the generation of junctional sequences has proceeded rapidly in recent years (2, 3), the mechanism of hypermutation continues to remain enigmatic.Current evidence suggests that hypermutation is induced during an immune response (4-6), introduces mainly singlenucleotide replacements at a rate estimated to be 10 .3 per base pair per cell division (7), acts efficiently only in and immediately around fully rearranged V genes (8-14), and is not causally linked to isotype switching (15, 16). Since V gene hypermutation plays a central role in the affinity maturation of antibodies (6,17,18), and is intimately associated with the development of B cell memory (17,(19)(20)(21)(22)(23), the complete elucidation of its mechanism, as well as how this mechanism is regulated, is of central importance to an understanding of the development of antibody specificity and humoral immunity.While direct approaches to the cha...

Increased Transcription Levels Induce Higher Mutation Rates in a Hypermutating Cell Line

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

“…The requirement of transcription-related sequences (Azuma et al 1993;Betz et al 1994;Giusti & Manser 1994;Bachl & Wabl 1996;Peters & Storb 1996) but dispensability of the variable gene sequence (Yelamos et al 1995) suggests that transcription elements recruit the machinery to the locus, while secondary structure enhances local hypermutability. The evidence connecting gene conversion to somatic hypermutation, as well as direct evidence from hypermutating cell lines with constitutive expression of terminal deoxynucleotidyl transferase (TdT) suggests that breaks are a feature of this mechanism (Sale & Neuberger 1998).…”

A Pivotal Role for DNase I-Sensitive Regions 3b and/or 4 in the Induction of Somatic Hypermutation of IgH Genes

“…26). Somatic hypermutation was detected in CAT but not in the V H promoter or E/MAR flanking it, although the frequency of mutation was approximately 1/10 that observed in endogenous V H -D-J H , suggesting that these cis-acting elements were critical for the induction of hypermutation and that other components such as C␥, C␣, or the enhancer flanking 3Ј of C␣ (3ЈE) (27)(28)(29) might be responsible for high-frequency somatic hypermutation (19,20,30).…”