Multigene Organization of Immunoglobulin Genes
Recall that Ig proteins consist of two identical heavy chains and two identical light chains (see Chapter 3). The light chains can be either kappa (κ) light chains or lambda (λ) light chains. The heavy-chain, κ, and λ gene families are each encoded on separate chromosomes. The relative locations (or loci; singular, locus) of the various gene segments are illustrated in Figure 6-6a.
FIGURE 6-6 Organization of immunoglobulin germ-line gene segments in the mouse. (a) The κ and λ light chains are encoded by V, J, and C gene segments. The heavy chain is encoded by V, D, J, and C gene segments. (b) The κ, λ and H-chain gene segments in the mouse genome are illustrated in such a way as to indicate both the large numbers of V gene segments and the overall size of each of the V gene loci. Note the scale bars that describe the overall size of each locus. [Data from Jhunjhunwala, S., M. C. van Zelm, M. M. Peak, and C. Murre. 2009. Chromatin architecture and the generation of antigen receptor diversity. Cell 138:435; Martinez-Jean, C., G. Folch, and M. -P. LeFranc. 2001. Nomenclature and overview of the mouse (Mus musculus and Mus sp.) immunoglobulin kappa (IGK) genes. Experimental and Clinical Immunogenetics 18:255; and Lefranc, M.-P. and G. Lefranc. Immunoglobulin lambda (IGL) genes of human and mouse, in: Molecular Biology of B cells (Honjo, T., F. W. Alt, and M. S. Neuberger, eds). Academic Press, Elsevier Science, pp. 37–59 (2004).]
κ Light-Chain Genes Include V, J, and C Segments
The mouse immunoglobulin κ light-chain locus (Igκ) spans approximately 3.2 megabases (Mb) and, depending on the mouse strain, includes 120–140 Vκ genes of which about 94–96 are functionally capable of encoding a Vκ protein segment (Figure 6-6b). Note that the precise numbers of gene segments vary according to the strain in mice and the individual in humans, and so these numbers must be regarded as approximations. Individual Vκ segments in both species are separated by noncoding gaps of 5 to 100 kb.
The transcriptional orientation of particular Vκ segments may be in the same or in the opposite direction as the constant region segment. The relative orientations of the variable and constant region segments do not affect the frequency of use of the segments, but do alter some details of the recombinational mechanism that creates the complete light-chain gene, as will be discussed later.
Downstream of the mouse Vκ region cluster are four functional Jκ segments and one pseudo-Jκ segment gene that contains a stop codon and therefore cannot be used. A similar arrangement is found in the human Vκ locus, although the number of functional Vκ gene segments in humans, 41, is a little lower than has been found in mice (Table 6-1). A single Cκ segment is found downstream of the J region, and all κ light-chain constant regions are encoded by this segment.
| Nature of segment | Number of heavy-chain segments (estimated) | Number of κ-chain segments (estimated) | Number of λ-chain segments (estimated) |
|---|---|---|---|
|
V |
45 |
41 |
33 |
|
D |
23 |
||
|
J |
6 |
5 |
5 |
|
Possible number of combinations |
45 × 23 × 6 = 6210 |
41 × 5 = 205 |
33 × 5 = 165 |
|
Possible number of heavy- and light-chain combinations in the human = 6210 × (205 + 165) = 2.3 × 106 |
|||
λ Light-Chain Genes Include Paired J and C Segments
The mouse immunoglobulin λ light-chain locus (Igλ) spans a much smaller region of approximately 240 kb (note the scale in Figure 6-6b). λ light chains are found in only 5% of mouse antibodies because of a deletion event in the mouse genome that has eliminated most of the λ variable gene segments. It was therefore not surprising to discover that there are only three fully functional Vλ gene segments in laboratory mice and just a few more in wild mice. Surprisingly, there are also three fully functional λ-chain constant regions, each one associated with its own J region segment (see Figure 6-6a). The Jλ4-Cλ4 segments are not expressed as proteins because of an RNA splice site defect.
Since recombination of Ig gene segments always occurs in the downstream direction (V to J), the location of the Vλ1 variable region sequence upstream of the Jλ3-Cλ3 and Jλ1-Cλ1 segments but downstream from the Jλ2-Cλ2 segments means that Vλ1 is always expressed with either Jλ3-Cλ3 or Jλ1-Cλ1 but never with Jλ2-Cλ2. For the same reason, Vλ2 is usually associated with Jλ2-Cλ2, although occasional recombination of Vλ2 with the 190-kb distant Jλ1-Cλ1 segment has been observed.
In humans, 40% of light chains are of the λ type, and about 33 Vλ-chain gene segments are used in mature antibody light chains. Downstream from the human Vλ locus is a series of seven Jλ-Cλ pairs, of which four or five pairs are fully functional.
Heavy-Chain Gene Organization Includes VH, D, JH, and CH Segments
Multiple VH gene segments are located across a region of approximately 2.4 Mb in mice (see Figure 6-6b). Mice express approximately 100 different VH segments, all of which are arranged in the same transcriptional orientation. At a distance 100 kb downstream from the cluster of mouse VH region segments is an 80-kb region containing approximately 14 D segments. Just 0.7 kb downstream of the most 3′ D segment is the JH region cluster, which contains four functional JH regions. A further gap separates the last JH segment from the first constant region exon encoding Cμ1. Within the 100-kb gap between the VH and DH regions is a critical regulatory element termed the intergenic control region 1 (IGCR1). The IGCR1 suppresses VH transcription and rearrangement at the DH-to-JH joining stage (see below).
The human VH locus has a similar arrangement with approximately 45 functional VH segments, 23 functional D segments, and 6 functional JH segments. Some human D regions can be read in all three reading frames, whereas mouse D regions are read mainly in reading frame 1, because of the presence of stop codons in their reading frames 2 and 3.
The eight constant regions of antibody heavy chains are encoded in a span of 200 kb of DNA downstream from the JH locus in the order μ, δ, γ3, γ1, γ2b, γ2a, ε, α. Recall that the constant regions of antibodies determine their heavy-chain class and, ultimately, their effector functions (see Chapters 3 and 12).
The Antibody Genes Found in Mature B Cells Are the Product of DNA Recombination
By now, it should be clear that the puzzle of the enormous diversity of the antibody repertoire is at least partially solved by the ability of developing B cells to recombine the V, D, and J gene segments that encode the heavy and light-chain variable regions in many different combinations in developing B cells. Specifically, each B cell uses one Vκ or one Vλ coupled with one Jκ or one Jλ, respectively, to create a single light-chain variable region gene. It also recombines one VH, one D, and one JH segment to form a heavy-chain variable region gene. As we will see below, the large number of different combinations of V and J segments for light chains and of V, D, and J segments for heavy chains provides a major source of the diversity in variable regions that determines antigen-binding specificity. Clues about some of the additional mechanisms of generating diversity in antigen-binding regions of antibodies came directly from studies of the mechanism of V(D)J recombination.