At least two types of somatic recombination are necessary for the generation of a complete immunoglobulin gamma 2b gene from germ-line DNA sequences. The first type of recombination consists of the assembly of three separate DNA segments, each encoding a different part of the variable region. The second type of recombination replaces the exons coding for the constant region of the mu chain with those coding for the same region of the gamma 2b chain. The DNA sequencing studies suggest that the two types of recombination operate by different mechanisms.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here, we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2, and H13, using the enhanced avidity of its IgG to overcome lower affinity Fab binding, as observed with other receptor-binding site antibodies. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small molecule therapeutics.

Complete, active, immunoglobulin variable (V) 1 genes are generated by somatic rearrangement of DNA sequences during the differentiation of lymphocyte precursor cells (1, 2). In the mouse, the conventionally defined V region of the light chains, both and ~¢ types, consists of two DNA segments: VL and Je, which are separate in the germline genome. The two DNA segments are joined at the 3' end of the V DNA and the 5' end of the J DNA to create a complete, light chain V gene (3)(4)(5)(6)(7)(8). In contrast, the heavy chain variable gene is encoded by three DNA segments, Vn, diversity (D) (which encodes primarily the third hypervariable region) (9), and JH. Both VH-D and D-JH joinings are necessary to generate a complete, heavy chain V gene (10,11).Sequencing studies revealed the presence of two blocks of highly conserved sequences around the recombination sites. In the 5' flanking region of the J DNAs, a palindromic heptamer, CACTGTG, and a T-rich nonamer, GGTTTTTGT, are conserved, and the sequences complementary to the heptamer and nonamer are ubiquitous in the 3' flanking region of the germline V DNA segments. It has been proposed that these two blocks of sequences constitute a recognition signal for the putative recombinase (7,8). The lengths of the spacers between the heptamer and the nonamer are strikingly regular (10, 11). The V~ and J~ DNA segments have spacers of 23 and 12 base pairs (bp), respectively (3, 12). In contrast, the Vx and Jg spacers are 12 ± 1 and 23 ± 1 bp, respectively (7,8). In addition, the spacers of all VH and JHare 23 ± 1 bp (10,11,(13)(14)(15).We and others proposed that the putative recombinase for V-J and V-D-J joinings contains two DNA-binding proteins: one recognizing the signal sequences with a shorter (12 bp) spacer and the other with a longer (23 bp) spacer (12/23-bp spacer rule), and all recombinations leading to the generation of a complete V gene, namely, Vt¢ and J~, VJk and J~, VH and D, and D and JH joinings, are mediated by the same or similar enzymes following this rule (10,11). This proposal predicted that the germline D segment would carry the signal sequences with a shorter spacer on each side. This prediction was met by our recent study: two DNA segments identified on the germline genome carried the conserved sequences with a 12-bp spacer on each side (16,17). We now describe the structure and organization of more germline D segments and present a model explaining how these D segments might become part of the complete VH gene active in myelomas during differentiation of lymphocytes.

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A putative diversity segment of immunoglobulin heavy-chain genes (D segments) has been identified 700 base pairs 5' to JH1 DNA on the germ-line genome of the mouse. This 10-base pair D segment is flanked by two sets of sequences related to (SEE FORMULAR IN TEXT) which are possible recognition sites for a recombinase. The spacer separating the heptamer and the nonamer is 12 base pairs long on both sides of the D segment. As the space separating the two signal sequences in VH DNAs and JH DNAs is 23 +/- 1 base pairs long, the two recombinations required for creation of a complete immunoglobulin VH gene, a VH--D joining and a D--JH joining, follow a 12/23-base pair spacer rule. Allelic exclusion is discussed with respect to D segments.