During the vertebrate immune response, Ig and TCR play central roles in antigen recognition. The NH2
-terminal portion of their subunits is called the V region because of its diverse amino acid sequence required for interaction with a diverse spectrum of antigens. Generation of the primary V-region repertoire depends on the common genetic basis and molecular mechanisms characteristic of these antigen-receptor molecules (1
). First, the V regions are encoded by two or three genetic segments, namely V, D, and J segments, each of which comprises multiple copies and provides the repertoire before somatic mutation. Second, during the ontogeny of lymphocytes, each one of these segments is chosen to undergo a somatic recombination event called V-(D)-J recombination, giving rise to the combinatorial and junctional amino acid diversity. Upon encounter with antigens, further diversification and refinement of the Ig repertoire is accomplished by a process known as affinity maturation, which includes somatic hypermutation, receptor editing, somatic gene conversion, and clonal selection (1
). In contrast, the V-region diversity of TCR is fixed through the selection process in the thymus and maintained without any modification (3
). Although these molecules are likely to be derived from a common ancestral receptor molecule, much more complex molecular mechanisms are used for the refinement of the V-region repertoire of Ig than TCR after maturation of lymphocytes.
The Ig molecule is encoded by three independent gene loci, namely Igκ and Igλ genes for the L chain and IgH genes for the H chain, which are located on chromosome 2 (4
), chromosome 22 (5
), and chromosome 14 (7
), respectively. Each of these loci spans a large DNA region of from one to a few megabases (Mb)1
). Although antibody function is determined by the complementation of L and H chains, accumulating evidence suggests that the major contribution to the generation of the diversity and specificity of Ig is from the H-chain molecule. Existence of an additional set of gene segments, namely D segments, and their involvement in V-D-J recombination increases enormously the sequence variability of the VH
region. Receptor editing by rearrangement of the silent allele has not been reported for the H-chain locus (13
), possibly indicating a critical role of the H chain for the antigen specificity of the Ig molecule.
It is, therefore, important to have the complete structure of the human VH
locus in order to understand the origin and behavior of the human immune repertoire. In addition, such studies will be useful in designing humanized antibodies. One of the best examples is the establishment and analysis of the xenomouse, which has deletions of the endogenous JH
and Jκ loci but carries human VH
and Vκ segments as transgenes to produce known human antibodies (15
). Knowledge of the number and organization of germline VH
and Vκ segments is essential to test the correlation between the germline repertoire and B lymphocyte repertoire formation in vivo.
Comparison of nucleotide sequences of the 5′-regulatory region of VH segments may tell us how human VH segments are transcriptionally regulated. Because the recombination signal sequences (RSS) flanking the germline gene segments play a key role in V-D-J rearrangement, it is interesting to test the correlation between the usage of individual VH segments and the sequence variation within the RSS. Existence of a novel VH family may provide additional V-region diversity. Isolation of polymorphic markers along the locus will greatly facilitate IgH haplotyping and subsequent systematic genetic analyses to examine the possible association between polymorphisms of the VH locus and susceptibility to immune disorders. It is also feasible to search for somatic gene conversions that have not yet been demonstrated in humans, the most critical test of which would be the extensive sequence comparison between germline and rearranged VH segments.
From an evolutionary point of view, nucleotide sequence comparison between different parts of the locus will enable us to trace evolution of this multigene family by DNA reorganization. It would also be very interesting to clarify the origin and nature of the translocated VH
loci on chromosomes 15 and 16 (16
). The existence of many VH
pseudogenes suggests frequent gene conversions during the evolution of the VH
). Moreover, comparative analysis of the structure and organization of the human VH
locus with those of other species or with other multigene loci (Vκ, Vλ, and TCR) will provide clues for further understanding the molecular mechanisms that govern the evolution of multigene families. Finally, the VH
locus that lies adjacent to the 14q telomere may provide a suitable candidate for the study of the structure of a human telomere.
To address the above questions, earlier studies on the organization of the human VH
locus have resulted in the completion of the physical map of the entire locus by isolation of yeast artificial chromosome (YAC) clones (8
). Here, we report the determination of the complete nucleotide sequence of the 957-kb DNA encompassing the human VH
locus consisting of 123 VH
segments. This permitted the classification of the VH
segments according to their structure and utilization into 39 functional, 1 transcribed, 4 open reading frame (ORF), and 79 pseudogenes. Both frequent DNA reorganization after mammalian divergence and high levels of repetitive elements were revealed. We also identified a putative ancestral VH
segment that is distantly related to VH
segments of other vertebrates as well as to those of humans. A single exon-encoded nonimmunoglobulin gene of unknown function was mapped in the JH