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Chapter 7 : Molecular Genetics of Antibody Diversity

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Abstract:

The vast diversity of the antibodies generated by B cells derives largely from the fact that the variable portion of these molecules is assembled differently in each individual lymphocyte using a large number of individual genes. Three types of genes known as the variable (V), diversity (D), and joining (J) genes need to be rearranged for the H-chain variable region to be complete, while only two of them, V and J, are used for the L-chain variable region. Preceding each V gene is a small exon encoding a hydrophobic leader sequence (Ls) that is responsible for guiding the nascent H-chain or L-chain protein into the lumen of the endoplasmic reticulum for processing and assembly. The Immunoglobulin H-Chain variable (IGHV) genes have been divided into 14 to 15 subgroups labeled IGHV1 to IGHV15. Subgroups are formed from closely related IGHV genes, but the related genes are not necessarily grouped together on the chromosome. It should be noted, however, that this total change in nucleotide number is ultimately affected not only by P-nucleotide addition but also by the processes of junctional flexibility and N-nucleotide addition that further contribute to antibody diversity. One of the most perplexing puzzles in the molecular genetics of antibody production was the ability of an animal to produce different isotype antibodies of identical antigenic specificity. There are numerous regulatory processes that control B-cell development, including transcriptional regulation of the immunoglobulin genes, allelic exclusion, and sequential rearrangement of L-chain variable-region genes.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7

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Figures

Image of Figure 7.1
Figure 7.1

Early theories of antibody production. Ehrlich's side-chain hypothesis. Ehrlich proposed that B cells possess on their surface a number of different side chains (analogous to antigen receptors) formed by the B cell before its encounter with antigen, each with the ability to bind to a unique antigen. Binding of a foreign body to one side chain would trigger overproduction of and secretion of that particular side chain. The instructive hypothesis states that a nascent antibody is a “blank template” with no antigenic specificity at all. Upon its first encounter with antigen, the antibody adopts a conformation permitting specific binding to that particular antigen.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.2
Figure 7.2

Dreyer and Bennett's “two genes, one polypeptide” theory departed from dogma, stating that a single antibody protein chain is produced from two genes. This theory was created to explain the duality of antibody structure (i.e., one portion of an antibody is fairly constant, while another portion is highly variable). According to this theory, during antibody production a gene encoding the highly variable (antigen-binding) portion of the antibody would be joined with the gene encoding the constant region of the antibody. RI and dIII indicate the positions of the restriction endonuclease recognition sites. Note that the size distribution of restriction fragments after rearrangement differs from that of the same locus before rearrangement. In the example shown, the two dIII restriction sites are lost following rearrangement of the DNA to form a functional gene encoding both the variable and constant regions of one antibody chain.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.3
Figure 7.3

Schematic diagram of the experiments of Tonegawa and Hozumi that demonstrated for the first time that production of antibody genes is linked to a physical rearrangement of genomic DNA. DNA from B-cell-like myeloma cells and from non-antibody-producing embryonic cells was digested with restriction endonuclease and the fragments separated by size and then analyzed with a probe that would specifically detect the antibody κ L-chain gene. Tonegawa and Hozumi found that whereas the κ L-chain gene was present on two DNA fragments in non-immunoglobulin-producing cells, the κ-chain gene was present on only one DNA fragment of a unique size in antibody-secreting myeloma cells. This indicates that the antibody-producing cells have DNA rearrangements at or near the antibody genes.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.4
Figure 7.4

Genomic organization of the mouse IGH-chain locus. The 5΄ end of the mouse IGH locus contains genes that are rearranged to form the variable-domain exon. The IGH-chain locus contains three different types of genes to form the complete variable-region gene: IGHV (variable), IGHD (diversity), and IGHJ (joining). In the mouse locus, there are between 100 and 1,000 IGHV genes (the actual number varies among mouse strains and is not fully known at this time). These genes are organized into groups designated IGHV1 to IGHV15, but individual genes are interspersed throughout the IGHV locus. Located upstream of the IGHV region is a cluster of 13 IGHD genes with no ordered nomenclature. The four IGHJ genes lie downstream of the IGHD genes. Each individual IGHV gene is preceded by a leader exon that encodes the hydrophobic leader sequence that guides nascent immunoglobulin proteins into the endoplasmic reticulum; each leader exon has its own promoter. The 3΄ end of the IGH-chain locus (starting about 8 kb downstream of the IGHJ genes) contains the genes encoding the constant regions of all of the different isotypes of antibody-IGHC. In the mouse genome, they are arranged sequentially: IGHM, IGHD, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, IGHA. It should be noted that each constant-region gene is depicted here as a single box for simplicity. () In reality, each of these constant- region genes is composed of a number of exons separated from each other by intronic sequence. The IGHM-chain gene and the IGHG3 gene are shown as examples. The IGHM chain lacks a hinge region; therefore, its constant gene consists of four exons (CM1 to CM4), each encoding one constant domain. In contrast, the H chain of IGG3 contains a hinge region. Its constant gene contains three exons (CG3-1 to CG3-3) encoding the three constant-region domains and four smaller exons, which together encode the flexible hinge region. Chromosomal location and basic organization of the human IGH locus. Located on the long arm of chromosome 14 there are 123 to 129 IGHV, 27 IGHD, 9 IGHJ, and 11 IGHC genes, but not all of the IGHV, IGHD, and IGHJ genes are functional. From IMGT, http://imgt.cines.fr, with permission from M.-P. Lefranc.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.5
Figure 7.5

The mouse IGK locus on chromosome 6. A total of 158 IGKV genes belonging to 19 subgroups extending over on 3,200 kb of DNA have been identified. Of the IGKV genes, 93 are functional (green), 6 form ORFs that could potentially encode a gene (yellow), and 59 are pseudogenes (red). The known repertoire is 93 established functional IGKV genes belonging to 18 subgroups. ORFs and currently unmapped IGKV genes may change the final number of functional IGKV genes. There are five IGKJ genes (orange) of which one, IGKJ3, is not functional, and one IGKC gene (blue). Genes unrelated to IGK are open boxes. Enhancer elements 5΄ and 3΄ (E5΄ and E3΄) of the IGKC gene are small circles. From IMGT, http://imgt.cines.fr, with permission from M.-P. Lefranc.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.6
Figure 7.6

Schematic representation of the genomic organization of the human κ L-chain locus on chromosome 2. The human IGK locus contains two IGKV-region clusters separated by 800 kb of intervening DNA: the distal cluster containing 36 IGKV genes that is at the 5΄ end of the locus and the proximal cluster containing 40 IGKV genes that is at the 3΄ end of the locus (shown above the distal cluster). There are five functional human IGKJ genes and a single IGKC gene. The IGKV genes that make up the proximal cluster are designated by a number for the family subgroup of which the gene is a member, followed by a hyphen and a number for where the gene is located in the locus relative to the IGKC-region gene. Smaller numbers are located closer to the IGKC gene. The IGKV genes of the distal cluster have the same numbers as the corresponding genes in the proximal cluster but are further identified with the letter D added. In some cases there are deletions in the clusters; these are indicated by small triangles. Functional genes are shown in green, pseudogenes are red, and open reading frames with the potential to encode a functional gene are yellow. The IGKJ gene cluster is orange, and the IGKC gene is blue. Map of the localization of human IGK genes on chromosome 2. From IMGT, http://imgt.cines.fr, with permission from M.-P. Lefranc.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.7
Figure 7.7

The immunoglobulin λ-chain locus. The murine IGL locus on chromosome 16 covers 200 kb and is organized into two duplicated units. The first unit contains two functional IGLV genes (green) and two IGLJ and IGLC genes, of which only one pair is functional (filled yellow and blue; nonfunctional genes in yellow-bordered and open blue box). The second duplicated unit has one IGLV gene, two functional IGLJ genes, two functional IGLC genes, and one IGLJ pseudogene (IGLJ3P). Rearrangement of the mouse immunoglobulin λ locus involves the joining of a single IGLV gene with one or two of the IGLJ genes that lie within the same duplicated unit as the IGLV gene. Thus, only four combinations are possible. For example, IGLV2 and IGLV3 can only recombine with IGLJ2 and IGLC2. Due to the presence of three functional IGLC genes, mouse IGL chains have three different isotypes. The human IGL locus is on chromosome 22 and extends over 1,050 kb. As depicted as a chromosomal representation, there are 70 to 71 IGLV-region genes and 7 to 11 pairs of IGLJ/IGLC genes. Twenty-nine to 33 of the IGLV-region genes belonging to 10 subgroups are functional. Four to five of the IGLJ/IGLC-region genes are functional. From IMGT, http://imgt.cines.fr, with permission from M.-P. Lefranc.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.8
Figure 7.8

V(D)J recombination is precisely targeted to IGV, IGD, and IGJ segments by RSSs that flank each gene in the germ line DNA. An RSS consists of three elements: a palindromic heptamer, an AT-rich nonamer, and a spacer. The nucleotide sequences of the heptamer and nonamer are critical for V(D)J recombination to occur, while the length of the spacer (but not its nucleotide sequence) is important. Depending on the spacer length, RSSs exist in two varieties, called 12-bp and 23-bp. The blue triangle represents the 12-bp RSS, and the yellow triangle represents the 23-bp RSS.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.9
Figure 7.9

Arrangement of 12-bp and 23-bp RSSs in genes of immunoglobulin loci. In the IGH locus, IGHV (V) and IGHJ (J) genes are flanked 5΄ and 3΄, respectively, by a 23-bp spacer RSS, and IGHD (D) genes are flanked by a 12-bp spacer RSS on both sides. The 12/23 rule ensures that an IGHD gene can recombine only with an IGHV gene or an IGHJ gene, not with another IGHD gene. In the IGK locus, the IGKV genes are flanked 3΄ by 12-bp spacer RSSs while the IGKJ genes are flanked 5΄ by 23-bp spacer RSSs. In the IGL locus, the IGLV genes are flanked 3΄ by 23-bp RSSs while the IGLJ genes are flanked 5΄ by 12-bp spacer RSSs. In the case of both L chains, the 12/23 rule ensures that a V-region gene can recombine only with a J-region gene, not with another V-region gene.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.10
Figure 7.10

The relative orientation of genes in the germ line configuration determines the mechanism by which V(D)J recombination proceeds. During recombination of two genes (in this case, a κ-chain IGKV gene and a κ-chain IGKJ gene), the RSSs that flank the two genes are brought together in a in which the RSSs lie close together and are oriented in the same direction. The 3΄ end of the IGKV locus is joined to the 5΄ end of the IGKJ locus. If the two genes lie in the same transcriptional orientation (indicated by black arrows) in the germ line configuration, recombination must involve formation of a loop in the DNA. When the genes are recombined, the DNA sequence intervening between the two genes is permanently removed from the genome by a looping-deletion mechanism. Scissors indicate the action of an endonuclease at the boundary of the heptamer of the RSS. The thin arrow indicates joining of free DNA ends. If the two genes lie in opposite transcriptional orientations (indicated by black arrows), recombination requires only that the DNA bend back on itself to form the RSS synapse. Recombination in this case results not in the deletion of the intervening sequence but rather in its inversion (or “flipping”) within the genome. The blue arrowheads are included to emphasize the inversion of the intervening sequence. In this case the coding joint and signal joint remain on the chromosome with no DNA deleted.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.11
Figure 7.11

The recombination of V-region genes involves a number of steps. The first step is recognition of the RSSs by the complex, followed by the formation of a synapse. A single-stranded nick is introduced by the gene products followed by the ligation of signal ends to form a signal joint (red arrow), and the esterification reaction at the coding joint, which simultaneously produces a hairpin loop structure (blue arrows). The hairpin structure is cleaved to form single-stranded DNA overhangs (blue), which will lead to P-nucleotide addition. The overhang is used as a template by a DNA polymerase (POL), which synthesizes the complementary strand. This panel shows the hairpin loop at the end of the V gene as an example. The free DNA ends are trimmed, presumably by an exonuclease (represented by a pair of scissors). In some cases, during rearrangement of the H-chain, N-nucleotides are added by the enzyme TdT. The free DNA ends are then aligned polymerized by a DNA POL to fill existing gaps and the DNA double- strand break ligated together (LIG) to restore the integrity of the chromosome.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.12
Figure 7.12

Figure 7.12 Experimental use of an artificial recombination substrate to measure recombination. The substrate consists of a strong, constitutive promoter (e.g., one contained in a retroviral long terminal repeat [LTR]) and two genes, one that has a 12-bp RSS and the other that has a 23-bp RSS. The red zigzag arrow indicates the direction of transcription from the LTR promoter. The genes are oriented in the substrate such that they are recombined by inversional joining. Between the two genes is a selectable marker gene (in this example, the gene whose product creates resistance to the drug mycophenolic acid). The selectable marker is placed into the substrate in the wrong orientation to encode a functional protein . Inversional recombination results in flipping of the gene to the proper orientation and in production of the Gpt protein, conferring drug resistance . The red arrows represent the orientation of the promoter, and the light blue arrows represent the orientation of the gene. This recombination substrate can be used to isolate and identify genes encoding proteins that are essential for V(D)J recombination. The recombination substrate is stably transfected into a cell. This stable transfectant is then transiently transfected with a cDNA library. Transient transfectants that receive a gene encoding a recombinase (red cDNA) will recombine the artificial substrate, thus inverting the gene so that it is in the correct orientation to be expressed. Addition of mycophenolic acid to the transient transfectants allows selection of cells that received DNA (red) encoding the recombinase. The piece of DNA conferring resistance can be isolated, cloned, sequenced, and analyzed for protein structure and function.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.13
Figure 7.13

Transient recombination substrates are useful for studying the nucleotide sequences of coding and signal joints that are formed during V(D)J recombination and are therefore useful for ascertaining the contribution of P-nucleotide addition, N-nucleotide addition, and junctional flexibility to individual recombination events. The substrate (top of each panel) consists of a bacterial promoter (p) that drives expression of a selectable marker (in this example, the Chl gene, which confers resistance to the antibiotic chloramphenicol [Chl]). Between the promoter and the selectable marker is a transcriptional terminator (OOP) that is flanked by RSSs. The RSSs are oriented so recombination will result in deletional joining of the RSSs, removing the OOP and allowing expression of the Chl resistance marker. Depending on the original orientation of the RSSs in the recombination substrate, the substrate can be used to study the nucleotide sequences of coding joints or signal joints . The substrate also contains a bacterial origin of replication and a constitutive marker for selection in bacteria (e.g., one encoding ampicillin resistance; Amp) that allows cloning of DNA that has been recombined for analysis of the types of joining reactions that have taken place.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.14
Figure 7.14

The expression of TdT during B-cell development correlates with the incidence of N-nucleotide addition. In the mouse, the enzyme TdT is expressed during the pro-B-cell stage of maturation, when the immunoglobulin H chain is being rearranged. Accordingly, immunoglobulin H-chain coding joints contain N-nucleotides (in black text). By the time the B cell reaches the pre-B-cell stage, when L-chain rearrangement is occurring, TdT expression is shut off. Coding joints of the L-chain genes do not contain N-nucleotides, although they may still contain P-nucleotides (blue text).

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.15
Figure 7.15

Multiple genetic defects can adversely affect V(D)J recombination. The cloning of signal joints and coding joints from recombination events occurring in mutant B cells has demonstrated that B cells lacking functional RAG proteins are still capable of forming RSS synapses but fail to initiate V(D)J recombination, since single-stranded DNA nicks are not produced . Scissors represent an endonuclease. In contrast, B cells deficient in the DNA-PKcs enzyme initiate V(D)J recombination by forming RSS synapses and single-stranded DNA nicks but are impaired in generating joints correctly . A synopsis of DSBR enzymes that are required for V(D)J recombination. See text for details.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.16
Figure 7.16

The opening of hairpin loops at the coding joint can occur at different nucleotide positions. The examples shown depict opening of a hairpin loop at a variable gene (in green text) before its ligation to a diversity gene (in red text). If the opening of the loop (by the action of an endonuclease, represented by a pair of scissors) occurs at the same place where the loop formed, the gene remains unchanged from its original state. If the hairpin loop is opened asymmetrically, single-stranded DNA overhangs will be generated at the end of the gene. When filled in by DNA POL, these overhangs will become palindromic (P-nucleotide) additions (shown in blue text). The single-stranded overhangs can be two nucleotides or four nucleotides , depending on the location of the asymmetric cleavage.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.17
Figure 7.17

The removal or addition of individual nucleotides at the end of a gene (by P-nucleotide addition, junctional flexibility, or N-nucleotide addition) can enhance antibody diversity but also can cause RF shifts that can result in premature chain termination. The example shown depicts a V-D junction when 0 to 3 nucleotides are added or removed from the genes before formation of the coding joint. Blue letters indicate nucleotides added by the enzyme Tdt. The right-pointing arrow after some amino acid sequences indicates that translation continues beyond the point shown.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.18
Figure 7.18

Diagram of a rearranged variable-domain exon, highlighting the alignment of the V, D, and J genes with the codons encoding the three hypervariable loops (CDRs) of the domain. Note that CDRs 1 and 2 lie entirely within the IGHV gene, upstream of the IGHV-D and IGHD-J junctions. For this reason, these two CDRs are unaffected by junctional diversity. Only CDR3 is encoded by the IGHV-D and IGHD-J genes, and only CDR3 is diversified by the mechanisms of junctional flexibility, N-nucleotide addition, and P-nucleotide addition. P, nucleotides created as palindromic additions; N, nucleotides added by TdT.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.19
Figure 7.19

The frequency of mutations in SHM is dependent solely on the relative position of the immunoglobulin promoter and is not due to any quality unique to immunoglobulin genes. The nucleotide positions are shown on the horizontal axis in the bottom panel and indicate distance from the promoter.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.20
Figure 7.20

Affinity maturation is brought about by the random introduction of point mutations into immunoglobulin variable-domain exons (by SHM) followed by the selection of B cells with immunoglobulin mutations resulting in an increased affinity for antigen. After proliferation and SHM, the B cells become extremely susceptible to apoptosis and can survive only if they receive essential signals from FDCs located in the basal light zone of the germinal center. To receive these signals, the B cell must bind to antigen that is present as immune complexes (called iccosomes) on the membrane of the FDCs. Since the amount of antigen in the iccosomes is present in limiting quantities, the B cells must compete with each other for antigen binding. As a result, only those B cells with the highest affinity for antigen receive survival signals.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.21
Figure 7.21

Distribution of point mutations that accumulate after affinity maturation. The diagram shows the amino acid map of the variable H and variable L domains after an initial encounter with antigen (primary) and after a second or third (tertiary) encounter with the same antigen. For each encounter, the amino acid sequence is marked with arrowheads to indicate the location of point mutations. Note that the distribution of point mutations is confined largely to the CDRs of the variable domains and differs dramatically from that of mutations introduced by SHM ( Fig. 7.19 ). The reason for this difference is that the mutations shown are the combined result of SHM and affinity maturation. Mutations in the CDRs are selected for since they are most likely to result in an increase in antigen-binding affinity

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.22
Figure 7.22

Rearrangement of the mouse IGH- and IGL-chain loci occurs in a defined sequence, always beginning with the IGH-chain locus. IGH-chain rearrangement proceeds in two steps, the first being the juxtaposition of one IGHD gene and one IGHJ gene. The IGHD and IGHJ genes can be joined in any of three RFs. If the joining occurs in RF2, a truncated form of the H chain (called D) will be produced, which ultimately signals cell death. If the joining occurs in either RF1 or RF3, however, the IGHDJ gene will be joined to an IGHV gene. This step of maturation is marked by the appearance on the cell membrane of the pro-BCR, consisting of Ig-α, Ig-β, and calnexin. Successful rearrangement of the first IGH allele will inhibit rearrangement of the second IGH allele (allelic exclusion). If the first IGH allele is rearranged nonproductively, rearrangement will occur at the second IGH allele. If neither IGH allele is rearranged productively, the B cell dies by apoptosis. If one of the IGH alleles is rearranged productively, the immunoglobulin κ-chain (IGK) locus will become accessible to the recombination machinery. Note that the λ-chain locus is not rearranged at this time. Successful rearrangement of the first IGK allele will inhibit rearrangement of the second IGK allele (allelic exclusion). If the first IGK allele is rearranged nonproductively, rearrangement will occur at the second IGK allele. If one of the IGK alleles is rearranged productively, the recombination machinery is shut off and the B cell will produce an IgM/κ antibody. If neither IGK allele is rearranged productively, the λ-chain (IGL) locus will become accessible to the recombination machinery. Successful rearrangement of the first IGL allele will inhibit rearrangement of the second IGL allele by allelic exclusion. If the first IGL allele is rearranged nonproductively, rearrangement will occur at the second IGL allele. If one of the IGL alleles is rearranged productively, the recombination machinery is shut off and the B cell will produce an IgM/λ antibody. If neither IGL allele is rearranged productively, the B cell dies by apoptosis.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.23
Figure 7.23

mice have deficiencies of B cells and T cells because of an inability to carry out variable-region gene recombination for either immunoglobulin or T-cell receptor production . Blastocysts from mice can be rescued from this immunodeficient state by receiving an injection of embryonic stem (ES) cells . The targeted disruption of various genes in the ES cells prior to their being injected into the blastocyst is a useful way of assessing the role of the disrupted genes in B-cell or T-cell development . The resultant somatic chimera mice can be tested for the presence of mature B and T cells, and if none is found, the DNA of the mouse can be analyzed to see what gene was disrupted to prevent the ES cells from restoring B- or T-cell development.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.24
Figure 7.24

B-cell maturation. A diagram of the pre-BCR. In this complex, membrane-bound immunoglobulin H chain is associated with the surrogate L chain (comprising the proteins λ5 and VpreB) and the signaling components Ig-α and Ig-β. Schematic representation of B-cell development, highlighting status of IGH-chain and IGL-chain rearrangement and membrane expression of immunoglobulin, the B220 phosphatase, and CD43 glycoprotein. During the pro-B-cell stage, the cell can transiently express on its surface the pro-BCR. The pro-BCR consists of the proteins Ig-α, Ig-β, and calnexin and appears on the membrane of the B cell at the developmental stage where the immunoglobulin H chain is incompletely rearranged and has only completed IGHD-J rearrangement ( Fig. 7.22 ).

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.25
Figure 7.25

Genomic arrangements of the mouse immunoglobulin IGH locus IGK locus and IGL locus highlighting the locations of promoters (yellow arrows) and enhancers (green plus signs). The promoters preceding each V-region gene are all very weak and are unable to direct transcription in the absence of enhancer activity. In the genomic configuration (before IGHV-D-J rearrangement occurs), the promoters are too far away from the enhancers to benefit from the enhancers' activity (top, short red squiggly arrows). IGHV-D-J production brings the promoter close enough to the enhancers such that the latter can amplify transcription ( long red squiggly arrow).

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.26
Figure 7.26

Alternative usage of two poly(A) addition sites in the immunoglobulin H-chain constant (IGHC) region, which is accompanied by alternative RNA splicing, determines the selective production of either secreted or membrane-bound immunoglobulin. Use of a poly(A) site involves RNA cleavage at the site by an endonuclease and attachment of a poly(A) tail (AAAAA...) to the transcript. Utilization of the poly(A) site close to 3΄ exon IGHM4 (site 1) terminates the transcript at this place, removing the M1 and M2 exons from the mature transcript and allowing incorporation of amino acids that encode the secreted terminus of the IGHC. In contrast, utilization of the poly(A) site 3΄ of exon M2 (site 2) retains the M1 and M2 exons in the mature IGHC transcript, allowing production of the transmembrane form of the IGHC. Simultaneously, utilization of site 2 results in an alternative splicing scheme that removes from the transcript the hydrophilic secreted tail of the IGHC and includes in the transcript the M1 and M2 exons, which encode the hydrophobic transmembrane and hydrophilic cytoplasmic domains, respectively, of the membrane-bound form of the IGHC.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.27
Figure 7.27

A mechanism similar to that used to differentially produce secreted and membrane-bound immunoglobulin ( Fig. 7.26 ) is also responsible for the simultaneous production of IgM and IgD by a single B cell. A primary IGHC transcript contains the coding sequences necessary for the production of secreted IGHM, membrane-bound IgM, secreted IgD, and membrane-bound IgD. In the example shown, the utilization of polyadenylation [AAA(A)] site 4 allows the production of membrane-bound IgD. Note that this mechanism regulates the production of IgM and IgD, since all other isotypes are produced by class switch recombination, which requires rearrangement of DNA.

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Figure 7.28a

CS recombination is the means by which a B cell changes from production of an antibody of one isotype to production of an antibody of a different isotype but the same antigenic specificity. This is accomplished by the physical rearrangement of the IGHC locus that moves the IGHV-D-J gene from an initial location (immediately upstream of the exons encoding the constant region of IGHM) to a location immediately upstream of the exons encoding the constant region of another IGHC isotype. Switch regions serve as the -acting elements that target CS recombination to the appropriate location in the immunoglobulin locus and consist of tandem-repetitive sequences. A switch region is represented as an array of ellipses. During CS recombination, a recombination event causes a crossover of two switch regions belonging to different isotype exons. DNA sequences that lie between the original location of the IGHV-D-J gene and its final location are removed permanently from the genome by a looping deletion mechanism. Since the switch regions are tandem repeats, the synapse formed between the two switch regions can occur in one of several different registers, resulting in an imprecise recombination event. Since this recombination event happens in an intron, the imprecision of recombination has no effect on the translational reading frame. CS recombination at a given switch region is thought to be activated by the initiation of sterile transcription from a promoter (yellow arrow) located upstream of the switch region, at the so-called I exon. This sterile transcript (red zigzag arrow) is processed by polyadenylation and splicing (splicing joins the I exon to the constant-region exons), just like a normal productive transcript. This transcription may help make the chromatin structure at the switch region accessible to the recombinase machinery. The choice of isotype during CS recombination is governed in part by cytokines. The example shown depicts the action of interleukin- 4 (IL-4) in causing preferential class switching to the isotype IgE. IL-4 mediates this effect by inducing sterile transcription at the I exon promoter of the ε-chain switch region (S).

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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Image of Figure 7.28b
Figure 7.28b

CS recombination is the means by which a B cell changes from production of an antibody of one isotype to production of an antibody of a different isotype but the same antigenic specificity. This is accomplished by the physical rearrangement of the IGHC locus that moves the IGHV-D-J gene from an initial location (immediately upstream of the exons encoding the constant region of IGHM) to a location immediately upstream of the exons encoding the constant region of another IGHC isotype. Switch regions serve as the -acting elements that target CS recombination to the appropriate location in the immunoglobulin locus and consist of tandem-repetitive sequences. A switch region is represented as an array of ellipses. During CS recombination, a recombination event causes a crossover of two switch regions belonging to different isotype exons. DNA sequences that lie between the original location of the IGHV-D-J gene and its final location are removed permanently from the genome by a looping deletion mechanism. Since the switch regions are tandem repeats, the synapse formed between the two switch regions can occur in one of several different registers, resulting in an imprecise recombination event. Since this recombination event happens in an intron, the imprecision of recombination has no effect on the translational reading frame. CS recombination at a given switch region is thought to be activated by the initiation of sterile transcription from a promoter (yellow arrow) located upstream of the switch region, at the so-called I exon. This sterile transcript (red zigzag arrow) is processed by polyadenylation and splicing (splicing joins the I exon to the constant-region exons), just like a normal productive transcript. This transcription may help make the chromatin structure at the switch region accessible to the recombinase machinery. The choice of isotype during CS recombination is governed in part by cytokines. The example shown depicts the action of interleukin- 4 (IL-4) in causing preferential class switching to the isotype IgE. IL-4 mediates this effect by inducing sterile transcription at the I exon promoter of the ε-chain switch region (S).

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
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References

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1. Blunt, T.,, N. J. Finnie,, G. E. Taccioli,, G. C. M. Smith,, J. Demengeot,, T. M. Gottlieb,, R. Mizuta,, A. J. Varghese,, F.W. Alt,, P. A. Jeggo,, and S. P. Jackson. 1995. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine SCID mutation. Cell 80:813823.
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Tables

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Table 7.1

Correspondence of terminologies used to designate immunoglobulin genes and genetic elements

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
Generic image for table
Table 7.2

Chromosomal locations of the immunoglobulin H- and L-chain genes known to be used to produce functional immunoglobulin

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
Generic image for table
Table 7.3

Numbers of potentially functional V, D, and J genes in mouse and human immunoglobulin

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7
Generic image for table
Table 7.4

A comparison of V(D)J recombination and class switch recombination

Citation: Taccioli G. 2004. Molecular Genetics of Antibody Diversity, p 145-184. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch7

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