《免疫学》（英文版） Chapter 05 Organization and Expression of Immunoglobulin Genes

8536d_ch05_105-136 8/22/02 2: 46 PM Page 105 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immuno chapter 5 rganization and Expression of Vx JK Jx Immunoglobulin G enes RNA splicing NE OF THE MOST REMARKABLE FEATURES OF the vertebrate immune system is its ability to respond to an apparently limitless array of for eign antigens As immunoglobulin(Ig)sequence data accu- Genetic Model Compatible with Ig Structure mulated, virtually every antibody molecule studied was found to contain a unique amino acid sequence in its vari- n Multigene Organization of Ig Genes able region but only one of a limited number of invariant se Variable-Region Gene Rearrangements quences in its constant region. The genetic basis for this combination of constancy and tremendous variation in a Mechanism of Variable-Region DNA single protein molecule lies in the organization of the Rearrangements munoglobulin genes a Generation of Antibody Diversity In germ-line DNA, multiple gene segments encode por tions of a single immunoglobulin heavy or light chain. These a Class Switching among Constant-Region Genes gene segments are carried in the germ cells but cannot be a Expression of Ig Genes transcribed and translated into complete chains until they are rearranged into functional genes During B-cell matura a Synthesis, Assembly, and Secretion of tion in the bone marrow, certain of these gene segments are Immunoglobulins randomly shuffled by a dynamic genetic system capable of a Regulation of Ig.-Ge ene trans generating more than 10combinations.Subsequent processes increase the diversity of the repertoire of antibody a Antibody Genes and Antibody Engineering binding sites to a very large number that exceeds 10 by at least two or three orders of magnitude. The processes of B- cell development are carefully regulated the maturation of a progenitor B cell progresses through an ordered sequence of DNA. While we think of genomic DNA as a stable genetic Ig-gene rearrangements, coupled with modifications to the blueprint, the lymphocyte cell lineage does not retain an in- gene that contribute to the diversity of the final product. By tact copy of this blueprint. Genomic rearrangement is an es- the end of this process, a mature, immunocompetent B cell sential feature of lymphocyte differentiation, and no other will contain coding sequences for one functional heavy- vertebrate cell type t describes the detailed organization of has been shown to undergo this process hain variable-region and one light-chain variable-region his chapter fir The individual B cell is thus antigenically committed to a the immunoglobulin genes, the process of lg-gene rearrange- specific epitope. After antigenic stimulation of a mature B ment, and the mechanisms by which the dynamic im cell in peripheral lymphoid organs, further rearrangement munoglobulin genetic system generates more than 10 of constant-region gene segments can generate changes in different antigenic specificities. Then it describes the mecha the isotype expressed, which produce changes in the biolog- nism of class switching, the role of differential RNA process ical effector functions of the immunoglobulin molecule ing in the expression of immunoglobulin genes, and the without changing its specificity. Thus, mature B cells contain regulation of Ig-gene transcription. The chapter concludes chromosomal dNa that is no longer identical to germ-line plication of our knowledge of the molecular

chapter 5 DNA. While we think of genomic DNA as a stable genetic blueprint, the lymphocyte cell lineage does not retain an in￾tact copy of this blueprint. Genomic rearrangement is an es￾sential feature of lymphocyte differentiation, and no other vertebrate cell type has been shown to undergo this process. This chapter first describes the detailed organization of the immunoglobulin genes, the process of Ig-gene rearrange￾ment, and the mechanisms by which the dynamic im￾munoglobulin genetic system generates more than 108 different antigenic specificities. Then it describes the mecha￾nism of class switching, the role of differential RNA process￾ing in the expression of immunoglobulin genes, and the regulation of Ig-gene transcription. The chapter concludes with the application of our knowledge of the molecular Vκ Jκ Jκ 3′ Cκ VJ Cκ Polyadenylation RNA splicing (A)n 5′ L L ■ Genetic Model Compatible with Ig Structure ■ Multigene Organization of Ig Genes ■ Variable-Region Gene Rearrangements ■ Mechanism of Variable-Region DNA Rearrangements ■ Generation of Antibody Diversity ■ Class Switching among Constant-Region Genes ■ Expression of Ig Genes ■ Synthesis, Assembly, and Secretion of Immunoglobulins ■ Regulation of Ig-Gene Transcription ■ Antibody Genes and Antibody Engineering Organization and Expression of Immunoglobulin Genes O       the vertebrate immune system is its ability to respond to an apparently limitless array of for￾eign antigens. As immunoglobulin (Ig) sequence data accu￾mulated, virtually every antibody molecule studied was found to contain a unique amino acid sequence in its vari￾able region but only one of a limited number of invariant se￾quences in its constant region. The genetic basis for this combination of constancy and tremendous variation in a single protein molecule lies in the organization of the im￾munoglobulin genes. In germ-line DNA, multiple gene segments encode por￾tions of a single immunoglobulin heavy or light chain. These gene segments are carried in the germ cells but cannot be transcribed and translated into complete chains until they are rearranged into functional genes. During B-cell matura￾tion in the bone marrow, certain of these gene segments are randomly shuffled by a dynamic genetic system capable of generating more than 106 combinations. Subsequent processes increase the diversity of the repertoire of antibody binding sites to a very large number that exceeds 106 by at least two or three orders of magnitude. The processes of B￾cell development are carefully regulated: the maturation of a progenitor B cell progresses through an ordered sequence of Ig-gene rearrangements, coupled with modifications to the gene that contribute to the diversity of the final product. By the end of this process, a mature, immunocompetent B cell will contain coding sequences for one functional heavy￾chain variable-region and one light-chain variable-region. The individual B cell is thus antigenically committed to a specific epitope. After antigenic stimulation of a mature B cell in peripheral lymphoid organs, further rearrangement of constant-region gene segments can generate changes in the isotype expressed, which produce changes in the biolog￾ical effector functions of the immunoglobulin molecule without changing its specificity. Thus, mature B cells contain chromosomal DNA that is no longer identical to germ-line Kappa Light-Chain Gene Rearrangement 8536d_ch05_105-136 8/22/02 2:46 PM Page 105 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:

8536d_ch05_105-136 8/22/02 2: 46 PM Page 106 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 106 PART I1 Generation of B-Cell and T-Cell Response VISUALIZING CONCEPTS Hematopoictic stem cell None Lymphoid cel None Partial heavy-chain gene rearrangement Pro-B cell None Bone Complete heavy-chain gene rearrangement Pre-B cell u Heavy chain rogate light chain Light-chain gene rearrangement Immature b cell mIgM Change in RNA processing Mature b cell mlgM mlgD A Activated B cell x( Differentiation Periphera IgM-secretil 邮c③→米 Class switching secreting vanous Memory of various FIGURE5-1 Overview of B-cell development. The events that ripheral lymphoid organs require antigen. The labels mlgM and occur dur ng maturation in the bone marrow do not require anti- mlg D refer to membrane-associated Igs IgG, IgA, and lgE are se- gen, whereas activation and differentiation of mature B cells in pe. creted immunoglobulins logy of immunoglobulin genes to the engineering of anti- body molecules for therapeutic and research application Genetic Model Compatible Chapter II covers in detail the entire process of B-cell devel- with lg Structure opment from the first gene rearrangements in progenitor B cells to final differentiation into memory B cells and anti- The results of the immunoglobulin-sequencing studies de- body-secreting plasma cells. Figure 5-1 outlines the sequen- scribed in Chapter 4 revealed a number of features of tial stages in B-cell development, many of which result from immunoglobulin structure that were difficult to reconcile critical rearrangements with classic genetic models. Any viable model of the

biology of immunoglobulin genes to the engineering of anti￾body molecules for therapeutic and research applications. Chapter 11 covers in detail the entire process of B-cell devel￾opment from the first gene rearrangements in progenitor B cells to final differentiation into memory B cells and anti￾body-secreting plasma cells. Figure 5-1 outlines the sequen￾tial stages in B-cell development, many of which result from critical rearrangements. Genetic Model Compatible with Ig Structure The results of the immunoglobulin-sequencing studies de￾scribed in Chapter 4 revealed a number of features of immunoglobulin structure that were difficult to reconcile with classic genetic models. Any viable model of the 106 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS FIGURE 5-1 Overview of B-cell development. The events that occur during maturation in the bone marrow do not require anti￾gen, whereas activation and differentiation of mature B cells in pe￾ripheral lymphoid organs require antigen. The labels mIgM and mIgD refer to membrane-associated Igs. IgG, IgA, and IgE are se￾creted immunoglobulins. Lymphoid cell Partial heavy-chain gene rearrangement Hematopoietic stem cell Pro-B cell Complete heavy-chain gene rearrangement Pre-B cell Light-chain gene rearrangement Immature B cell Change in RNA processing Peripheral lymphoid organs Bone marrow Mature B cell Antigen stimulation Activated B cell Differentiation IgM-secreting plasma cells Class switching Memory B cells of various isotypes Plasma cells secreting various isotypes None CELL Ig EXPRESSED None None µ Heavy chain + surrogate light chain mIgM mIgM + mIgD IgM IgG IgA IgE 8536d_ch05_105-136 8/22/02 2:46 PM Page 106 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:

8536d_ch05_105-136 8/1/02 8: 53 AM Page 107 mac79 Mac 79:45_Bw Glasby et al. Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 107 immunoglobulin genes had to account for the following in rabbits by C. Todd, who found that a particular allotypic properties of antibodies marker in the heavy-chain variable region could be associ ated with a, Y, and u heavy-chain constant regions. Consid- The vast diversity of antibody specificities rable additional evidence has confirmed that a single The presence in Ig heavy and light chains of a variable ariable-region sequence, defining a particular antigenic region at the amino-terminal end and a constant region specificity, can be associated with multiple heavy-chain at the carboxyl-terminal end constant-region sequences; in other words, different classes, or isotypes, of antibody (e.g. IgG, IgM)can be expressed a The existence of isotypes with the same antigenic with identical variable-region sequences specificity, which result from the association of a given variable region with different heavy-chain constant region Dreyer and Bennett Proposed the Two-Gene Model Germ-Line and Somatic- Variation Models In an attempt to develop a genetic model consistent with the Contended To Explain Antibody Diversity known findings about the structure of immunoglobulins, w Dreyer and J. Bennett suggested, in their classic theoretical For several decades, immunologists sought to imagine a ge- paper of 1965, that two separate genes encode a single netic mechanism that could explain the tremendous diversity munoglobulin heavy or light chain, one gene for the V region of antibody structure. Two different sets of theories emerged. (variable region) and the other for the C region(constant re The germ-line theories maintained that the genome con- gion). They suggested that these two genes must somehow tributed by the germ cells, egg and sperm, contains a large come together at the dna level to form a continuous mes repertoire of immunoglobulin genes; thus, these theories in- sage that can be transcribed and translated into a single Ig voked no special genetic mechanisms to account for anti- heavy or light chain. Moreover, they proposed that hundreds body diversity. They argued that the immense survival value or thousands of V-region genes were carried in the germ line, of the immune system justified the dedication of a significant whereas only single copies of C-region class and subclass fraction of the genome to the coding of antibodies In con- genes need exist trast,the somatic-variation theories maintained that the The strength of this type of recombinational model genome contains a relatively small number of immunoglob- (which combined elements of the germ-line and somatic- ulin genes, from which a large number of antibody specifici- variation theories) was that it could account for those im ties are generated in the somatic cells by mutation or munoglobulins in which a single V region was combined recombination with various C regions. By postulating a single constant As the amino acid sequences of more and more im- region gene for each immunoglobulin class and subclass, the munoglobulins were determined, it became clear that there model also could account for the conservation of necessary sity but also for maintaining constancy. Whether diversity diversification of variable-region genes g for evolutionary must be mechanisms not only for generating antibody diver- biological effector functions while allowi was generated by germ-line or by somatic mechanisms, a At first, support for the Dreyer and Bennett hypothesis paradox remained: How could stability be maintained in the was indirect. Early studies of DNA hybridization kinetics us- constant(C)region while some kind of diversifying mecha- ing a radioactive constant-region DNA probe indicated that nism generated the variable(V)region? the probe hybridized with only one or two genes, confirming Neither the germ-line nor the somatic-variation propo- the models prediction that only one or two copies of each nents could offer a reasonable explanation for this central constant-region class and subclass gene existed. However, in- feature of immunoglobulin structure Germ-line proponents direct evidence was not enough to overcome stubborn resis- found it difficult to account for an evolutionary mechanism tance in the scientific community to the hypothesis of Dreyer that could generate diversity in the variable part of the many and Bennet. The suggestion that two genes encoded a single heavy-and light-chain genes while preserving the constant polypeptide contradicted the existing one gene-one region of each unchanged. Somatic-variation proponents polypeptide principle and was without precedent in any found it difficult to conceive of a mechanism that could di- known biological system. versify the variable region of a single heavy- or light-chain As so often is the case in science, theoretical and intellec gene in the somatic cells without allowing alteration in the tual understanding of lg-gene organization progressed ahead amino acid sequence encoded by the constant region. of the available methodology. Although the Dreyer and Ben- A third structural feature requiring an explanation nett model provided a theoretical framework for reconciling emerged when amino acid sequencing of the human the dilemma between Ig-sequence data and gene organiza myeloma protein called Til revealed that identical variable- tion, actual validation of their hypothesis had to wait for sev- region sequences were associated with both y and u heavy- eral major technological advances in the field of molecular chain constant regions. A similar phenomenon was observed biology

immunoglobulin genes had to account for the following properties of antibodies: ■ The vast diversity of antibody specificities ■ The presence in Ig heavy and light chains of a variable region at the amino-terminal end and a constant region at the carboxyl-terminal end ■ The existence of isotypes with the same antigenic specificity, which result from the association of a given variable region with different heavy-chain constant regions Germ-Line and Somatic-Variation Models Contended To Explain Antibody Diversity For several decades, immunologists sought to imagine a ge￾netic mechanism that could explain the tremendous diversity of antibody structure. Two different sets of theories emerged. The germ-line theories maintained that the genome con￾tributed by the germ cells, egg and sperm, contains a large repertoire of immunoglobulin genes; thus, these theories in￾voked no special genetic mechanisms to account for anti￾body diversity. They argued that the immense survival value of the immune system justified the dedication of a significant fraction of the genome to the coding of antibodies. In con￾trast, the somatic-variation theories maintained that the genome contains a relatively small number of immunoglob￾ulin genes, from which a large number of antibody specifici￾ties are generated in the somatic cells by mutation or recombination. As the amino acid sequences of more and more im￾munoglobulins were determined, it became clear that there must be mechanisms not only for generating antibody diver￾sity but also for maintaining constancy. Whether diversity was generated by germ-line or by somatic mechanisms, a paradox remained: How could stability be maintained in the constant (C) region while some kind of diversifying mecha￾nism generated the variable (V) region? Neither the germ-line nor the somatic-variation propo￾nents could offer a reasonable explanation for this central feature of immunoglobulin structure. Germ-line proponents found it difficult to account for an evolutionary mechanism that could generate diversity in the variable part of the many heavy- and light-chain genes while preserving the constant region of each unchanged. Somatic-variation proponents found it difficult to conceive of a mechanism that could di￾versify the variable region of a single heavy- or light-chain gene in the somatic cells without allowing alteration in the amino acid sequence encoded by the constant region. A third structural feature requiring an explanation emerged when amino acid sequencing of the human myeloma protein called Ti1 revealed that identical variable￾region sequences were associated with both and heavy￾chain constant regions. A similar phenomenon was observed in rabbits by C. Todd, who found that a particular allotypic marker in the heavy-chain variable region could be associ￾ated with , , and heavy-chain constant regions. Consid￾erable additional evidence has confirmed that a single variable-region sequence, defining a particular antigenic specificity, can be associated with multiple heavy-chain constant-region sequences; in other words, different classes, or isotypes, of antibody (e.g., IgG, IgM) can be expressed with identical variable-region sequences. Dreyer and Bennett Proposed the Two-Gene Model In an attempt to develop a genetic model consistent with the known findings about the structure of immunoglobulins, W. Dreyer and J. Bennett suggested, in their classic theoretical paper of 1965, that two separate genes encode a single im￾munoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant re￾gion). They suggested that these two genes must somehow come together at the DNA level to form a continuous mes￾sage that can be transcribed and translated into a single Ig heavy or light chain. Moreover, they proposed that hundreds or thousands of V-region genes were carried in the germ line, whereas only single copies of C-region class and subclass genes need exist. The strength of this type of recombinational model (which combined elements of the germ-line and somatic￾variation theories) was that it could account for those im￾munoglobulins in which a single V region was combined with various C regions. By postulating a single constant￾region gene for each immunoglobulin class and subclass, the model also could account for the conservation of necessary biological effector functions while allowing for evolutionary diversification of variable-region genes. At first, support for the Dreyer and Bennett hypothesis was indirect. Early studies of DNA hybridization kinetics us￾ing a radioactive constant-region DNA probe indicated that the probe hybridized with only one or two genes, confirming the model’s prediction that only one or two copies of each constant-region class and subclass gene existed. However, in￾direct evidence was not enough to overcome stubborn resis￾tance in the scientific community to the hypothesis of Dreyer and Bennet. The suggestion that two genes encoded a single polypeptide contradicted the existing one gene–one polypeptide principle and was without precedent in any known biological system. As so often is the case in science, theoretical and intellec￾tual understanding of Ig-gene organization progressed ahead of the available methodology. Although the Dreyer and Ben￾nett model provided a theoretical framework for reconciling the dilemma between Ig-sequence data and gene organiza￾tion, actual validation of their hypothesis had to wait for sev￾eral major technological advances in the field of molecular biology. Organization and Expression of Immunoglobulin Genes CHAPTER 5 107 8536d_ch05_105-136 8/1/02 8:53 AM Page 107 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:

8536d_ch05_105-136 8/22/02 2: 46 PM Page 108 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 108 PART I1 Generation of B-Cell and T-Cell Response Tonegawa's Bombshell--Immunoglobulin myeloma cells), the V and C genes undergo rearrangement. Genes Rearrange In the embryo, the V and C genes are separated by a large DNA segment that contains a restriction-endonuclease site; evidence that separate genes encode the V and C regions of together and the intervening DNA Sequence is eliminated In 1976, S. Tonegawa and N. Hozumi found the first direct during differentiation, the V and C genes are brought closer mmunoglobulins and that the genes are rearranged in the The pioneering experiments of Tonegawa and Hozumi course of B-cell differentiation. This work changed the field employed a tedious and time-consuming procedure that has of immunology. In 1987, Tonegawa was awarded the Nobel since been replaced by the much more powerful approach of Prize for this work. Southern-blot analysis. This method, now universally used to Selecting DNA from embryonic cells and adult myeloma investigate the rearrangement of immunoglobulin genes, cells-cells at widely different stages of development- eliminates the need to elute the separated DNA restriction Tonegawa and Hozumi used various restriction endonucle- fragments from gel slices prior to analysis by hybridization ases to generate DNA fragments. The fragments were then with an immunoglobulin gene segment probe Figure 5-2 separated by size and analyzed for their ability to hybridize shows the detection of rearrangement at the k light-chain lo- with a radiolabeled mRNA probe. Two separate restriction by cor fragments produced by digestion of fragments from the embryonic DNA hybridized with the DNA from a clone of B-lineage cells with the pattern ob- mRNA, whereas only a single restriction fragment of the tained by digestion of non-B cells(e. g, sperm or liver cells) adult myeloma DNA hybridized with the same probe. Tone- The rearrangement of a V gene deletes an extensive section of gawa and Hozumi suggested that, during differentiation of germ-line DNA, thereby creating differences between re- lymphocytes from the embryonic state to the fully differenti- arranged and unrearranged Ig loci in the distribution and ted plasma-cell stage (represented in their system by the number of restriction sites. This results in the generation of Germ line Rearranged Deleted ∥[c →5-作口[c}3 Rearrangement Probe Probe Re digestion RE digestion Germ line Southern FIGURE 5.2 Experimental basis for diagnosis of rearrangement at are separated in the germ line. Consequently, fragments dependent an immunoglobulin locus. The number and size of restriction frag. on the presence of this segment for their generation are absent from ments generated by the treatment of DNA with a restriction enzyme the restriction-enzyme digest of DNA from the rearranged locus. Fur is determined by the sequence of the DNA. The digestion of re- thermore, rearranged DNA gives rise to novel fragments that are ab ranged DNA with a restriction enzyme(RE) yields a pattern of re- sent from digests of DNa in the germ-line configuration. This can be striction fragments that differ from those obtained by digestion of an useful because both B cells and non-B cells have two immunoglob unrearranged locus with the same RE. Typically, the fragments are an- lin loci. One of these is rearranged and the other is not. Consequently, alyzed by the technique of Southern blotting. In this example, a probe unless a genetic accident has resulted in the loss of the germ-line lo- that includes a j gene segment is used to identify RE digestion frag. cus, digestion of DNA from a myeloma or normal B-cell clone will ments that include all or portions of this segment. As shown, re. produce a pattern of restriction that includes all of those in a germ- arrangement results in the deletion of a segment of germ-line dna line digest plus any novel fragments that are generated from the and the loss of the restriction sites that it includes. It also results in change in DNA sequence that accompanies rearrangement. Note the joining of gene segments, in this case a V and a segment, that that only one of the several gene segements present is shown

Tonegawa’s Bombshell—Immunoglobulin Genes Rearrange In 1976, S. Tonegawa and N. Hozumi found the first direct evidence that separate genes encode the V and C regions of immunoglobulins and that the genes are rearranged in the course of B-cell differentiation. This work changed the field of immunology. In 1987, Tonegawa was awarded the Nobel Prize for this work. Selecting DNA from embryonic cells and adult myeloma cells—cells at widely different stages of development— Tonegawa and Hozumi used various restriction endonucle￾ases to generate DNA fragments. The fragments were then separated by size and analyzed for their ability to hybridize with a radiolabeled mRNA probe. Two separate restriction fragments from the embryonic DNA hybridized with the mRNA, whereas only a single restriction fragment of the adult myeloma DNA hybridized with the same probe. Tone￾gawa and Hozumi suggested that, during differentiation of lymphocytes from the embryonic state to the fully differenti￾ated plasma-cell stage (represented in their system by the myeloma cells), the V and C genes undergo rearrangement. In the embryo, the V and C genes are separated by a large DNA segment that contains a restriction-endonuclease site; during differentiation, the V and C genes are brought closer together and the intervening DNA sequence is eliminated. The pioneering experiments of Tonegawa and Hozumi employed a tedious and time-consuming procedure that has since been replaced by the much more powerful approach of Southern-blot analysis. This method, now universally used to investigate the rearrangement of immunoglobulin genes, eliminates the need to elute the separated DNA restriction fragments from gel slices prior to analysis by hybridization with an immunoglobulin gene segment probe. Figure 5-2 shows the detection of rearrangement at the light-chain lo￾cus by comparing the fragments produced by digestion of DNA from a clone of B-lineage cells with the pattern ob￾tained by digestion of non-B cells (e.g., sperm or liver cells). The rearrangement of a V gene deletes an extensive section of germ-line DNA, thereby creating differences between re￾arranged and unrearranged Ig loci in the distribution and number of restriction sites. This results in the generation of 108 PART II Generation of B-Cell and T-Cell Responses FIGURE 5-2 Experimental basis for diagnosis of rearrangement at an immunoglobulin locus. The number and size of restriction frag￾ments generated by the treatment of DNA with a restriction enzyme is determined by the sequence of the DNA.The digestion of re￾arranged DNA with a restriction enzyme (RE) yields a pattern of re￾striction fragments that differ from those obtained by digestion of an unrearranged locus with the same RE. Typically, the fragments are an￾alyzed by the technique of Southern blotting. In this example, a probe that includes a J gene segment is used to identify RE digestion frag￾ments that include all or portions of this segment. As shown, re￾arrangement results in the deletion of a segment of germ-line DNA and the loss of the restriction sites that it includes. It also results in the joining of gene segments, in this case a V and a J segment, that are separated in the germ line. Consequently, fragments dependent on the presence of this segment for their generation are absent from the restriction-enzyme digest of DNA from the rearranged locus. Fur￾thermore, rearranged DNA gives rise to novel fragments that are ab￾sent from digests of DNA in the germ-line configuration. This can be useful because both B cells and non-B cells have two immunoglobu￾lin loci. One of these is rearranged and the other is not. Consequently, unless a genetic accident has resulted in the loss of the germ-line lo￾cus, digestion of DNA from a myeloma or normal B-cell clone will produce a pattern of restriction that includes all of those in a germ￾line digest plus any novel fragments that are generated from the change in DNA sequence that accompanies rearrangement. Note that only one of the several J gene segements present is shown. 5′ 3′ Vn RE V2 RE J C V1 RE RE RE 5′ 3′ Vn RE V2 RE V1 RE RE RE Germ line Rearranged Germ line Rearranged Deleted Rearrangement J C Probe Probe RE digestion RE digestion Southern blot 8536d_ch05_105-136 8/22/02 2:46 PM Page 108 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:

8536d_ch05_105-136 8/22/02 2: 46 PM Page 109 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 1 different restriction patterns by rearranged and unre- nucleotide sequence. When the nucleotide sequence was arranged loci. Extensive application of this approach has compared with the known amino acid sequence demonstrated that the Dreyer and Bennett two-gene chain variable region, an unusual discrepancy was observed. model-one gene encoding the variable region and another Although the first 97 amino acids of the A-chain variable re- encoding the constant region--applied to eavy and gion corresponded to the nucleotide codon sequence, the re- light-chain genes. nanning 13 carboxyl-terminal amino acids of the proteins ariable region did not. It turned out that many base pairs away a separate, 39-bp gene segment, called J for joining,en Multigene Organization of Ig Genes coded the remaining 13 amino acids of the A-chain variable region. Thus, a functional A variable-region gene contains As cloning and sequencing of the light- and heavy-chain two coding segments-a 5"'V segment and a 3 segment DNA was accomplished, even greater complexity was re- which are separated by a noncoding DNA sequence in unre- vealed than had been predicted by Dreyer and Bennett. The K arranged germ-line DNA rate multigene families situated on different chromosomes three Va gene segments, four Ja gene segments, and fouro and a light chains and the heavy chains are encoded by ser The A multigene family in the mouse germ line contain (Table 5-1. In germ-line DNA, each of these multigene fam- gene segments(Figure 5-3a). The JA4 is a pseudogene, a de ilies contains several coding sequences, called gene seg- fective gene that is incapable of encoding protein; such ments, separated by noncoding regions. During B-cell genes are indicated with the psi symbol (u). Interestingly, maturation, these gene segments are rearranged and brought J,4's constant region partner, CA4, is a perfectly functional together to form functional immunoglobulin genes. gene. The Va and the three functional Ja gene segments en- code the variable region of the light chain, and each of the Each Multigene Family Has Distinct Features three functional CA gene segments encodes the constant re gion of one of the three A-chain subtypes (Al, A2, and The K and A light-chain families contain V, J, and C gene seg- A3). In humans, the lambda locus is more complex. There ments; the rearranged V] segments encode the variable re- are 31 functional Va gene segments, 4 Jx segments, and gion of the light chains. The heavy-chain family contains V, 7 C, segments. In additional to the functional gene seg- D,J and C gene segments; the rearranged VD) gene seg- ments, the human lambda complex contains many VA, JA, ments encode the variable region of the heavy chain. In each and Cx pseudogenes. gene family, C gene segments encode the constant regions Each V gene segment is preceded at its 5 end by a small exon that encodes a short signal or leader (l) peptide that guides K-CHAIN MULTIGENE FAMILY the heavy or light chain through the endoplasmic reticulum. The K-chain multigene family in the mouse contains approx The signal peptide is cleaved from the nascent light and heavy imately 85 Vx gene segments, each with an adjacent leader se chains before assembly of the finished immunoglobulin mol- quence a short distance upstream(ie, on the 5 side). There ecule. Thus, amino acids encoded by this leader sequence do are five Jk gene segments(one of which is a nonfunctional not appear in the im dobulin molecule pseudogene)and a single Ck gene segment( Figure 5-3b).As the A multigene family, the Vx and Jk gene segments en A-CHAIN MULTIGENE FAMILY code the variable region of the K light chain, and the Ck gene The first evidence that the light-chain variable region was ac- segment encodes the constant region. Since there is only one gene segment, there are no subt abtypes of k light cha tually encoded by two gene segments appeared when Tone. Comparison of parts a and b of Figure 5-3 shows that the gawa cloned the germ-line DNA that encodes the variable region of mouse A light chain and determined its complet arrangement of the gene segments is quite different in the K and A gene families. The k-chain multigene family in hu mans,which has an organization similar to that of the mouse, contains approximately 40 k gene segments, 5 Chromosomal locations of segments, and a single CK segment. TABLE 5.1 immunoglobulin genes in human and mouse HEAVY-CHAIN MULTIGENE FAMILY The organization of the immunoglobulin heavy-chain genes CHROMOSOME is similar to, but more complex than, that of the k and Gene Human Mouse A light-chain genes( Figure 5-3c). An additional gene gment encodes part of the heavy-chain variable region λ Light chain The existence of this gene segment was first proposed by Leroy Hood and his colleagues, who compared the K Light chain heavy-chain variable-region amino acid sequence with the Heavy chain VH and JH nucleotide sequences. The VH gene segment was found to encode amino acids l to 94 and the JH gene segment

different restriction patterns by rearranged and unre￾arranged loci. Extensive application of this approach has demonstrated that the Dreyer and Bennett two-gene model—one gene encoding the variable region and another encoding the constant region—applied to both heavy and light-chain genes. Multigene Organization of Ig Genes As cloning and sequencing of the light- and heavy-chain DNA was accomplished, even greater complexity was re￾vealed than had been predicted by Dreyer and Bennett. The and light chains and the heavy chains are encoded by sepa￾rate multigene families situated on different chromosomes (Table 5-1). In germ-line DNA, each of these multigene fam￾ilies contains several coding sequences, called gene seg￾ments, separated by noncoding regions. During B-cell maturation, these gene segments are rearranged and brought together to form functional immunoglobulin genes. Each Multigene Family Has Distinct Features The and light-chain families contain V, J, and C gene seg￾ments; the rearranged VJ segments encode the variable re￾gion of the light chains. The heavy-chain family contains V, D, J, and C gene segments; the rearranged VDJ gene seg￾ments encode the variable region of the heavy chain. In each gene family, C gene segments encode the constant regions. Each V gene segment is preceded at its 5 end by a small exon that encodes a short signal or leader (L) peptide that guides the heavy or light chain through the endoplasmic reticulum. The signal peptide is cleaved from the nascent light and heavy chains before assembly of the finished immunoglobulin mol￾ecule. Thus, amino acids encoded by this leader sequence do not appear in the immunoglobulin molecule. -CHAIN MULTIGENE FAMILY The first evidence that the light-chain variable region was ac￾tually encoded by two gene segments appeared when Tone￾gawa cloned the germ-line DNA that encodes the variable region of mouse light chain and determined its complete nucleotide sequence. When the nucleotide sequence was compared with the known amino acid sequence of the - chain variable region, an unusual discrepancy was observed. Although the first 97 amino acids of the -chain variable re￾gion corresponded to the nucleotide codon sequence, the re￾maining 13 carboxyl-terminal amino acids of the protein’s variable region did not. It turned out that many base pairs away a separate, 39-bp gene segment, called J for joining, en￾coded the remaining 13 amino acids of the -chain variable region. Thus, a functional variable-region gene contains two coding segments—a 5 V segment and a 3 J segment— which are separated by a noncoding DNA sequence in unre￾arranged germ-line DNA. The multigene family in the mouse germ line contains three V gene segments, four J gene segments, and four C gene segments (Figure 5-3a). The J4 is a pseudogene, a de￾fective gene that is incapable of encoding protein; such genes are indicated with the psi symbol (). Interestingly, J4’s constant region partner, C4, is a perfectly functional gene. The V and the three functional J gene segments en￾code the variable region of the light chain, and each of the three functional C gene segments encodes the constant re￾gion of one of the three -chain subtypes (1, 2, and 3). In humans, the lambda locus is more complex. There are 31 functional V gene segments, 4 J segments, and 7 C segments. In additional to the functional gene seg￾ments, the human lambda complex contains many V, J, and C pseudogenes. -CHAIN MULTIGENE FAMILY The -chain multigene family in the mouse contains approx￾imately 85 V gene segments, each with an adjacent leader se￾quence a short distance upstream (i.e., on the 5 side). There are five J gene segments (one of which is a nonfunctional pseudogene) and a single C gene segment (Figure 5-3b). As in the multigene family, the V and J gene segments en￾code the variable region of the light chain, and the C gene segment encodes the constant region. Since there is only one C gene segment, there are no subtypes of light chains. Comparison of parts a and b of Figure 5-3 shows that the arrangement of the gene segments is quite different in the and gene families. The -chain multigene family in hu￾mans, which has an organization similar to that of the mouse, contains approximately 40 V gene segments, 5 J segments, and a single C segment. HEAVY-CHAIN MULTIGENE FAMILY The organization of the immunoglobulin heavy-chain genes is similar to, but more complex than, that of the and light-chain genes (Figure 5-3c). An additional gene segment encodes part of the heavy-chain variable region. The existence of this gene segment was first proposed by Leroy Hood and his colleagues, who compared the heavy-chain variable-region amino acid sequence with the VH and JH nucleotide sequences. The VH gene segment was found to encode amino acids 1 to 94 and the JH gene segment Organization and Expression of Immunoglobulin Genes CHAPTER 5 109 TABLE 5-1 Chromosomal locations of immunoglobulin genes in human and mouse CHROMOSOME Gene Human Mouse Light chain 22 16 Light chain 2 6 Heavy chain 14 12 8536d_ch05_105-136 8/22/02 2:46 PM Page 109 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e: