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 intact copy of this blueprint. Genomic rearrangement is an essential 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 rearrangement, and the mechanisms by which the dynamic immunoglobulin genetic system generates more than 108 different antigenic specificities. Then it describes the mechanism of class switching, the role of differential RNA processing 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 foreign antigens. As immunoglobulin (Ig) sequence data accumulated, virtually every antibody molecule studied was found to contain a unique amino acid sequence in its variable region but only one of a limited number of invariant sequences 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 immunoglobulin genes. In germ-line DNA, multiple gene segments encode portions 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 maturation 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 Bcell 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 heavychain 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 biological 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 antibody molecules for therapeutic and research applications. Chapter 11 covers in detail the entire process of B-cell development from the first gene rearrangements in progenitor B cells to final differentiation into memory B cells and antibody-secreting plasma cells. Figure 5-1 outlines the sequential 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 described 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 antigen, whereas activation and differentiation of mature B cells in peripheral lymphoid organs require antigen. The labels mIgM and mIgD refer to membrane-associated Igs. IgG, IgA, and IgE are secreted 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 genetic mechanism that could explain the tremendous diversity of antibody structure. Two different sets of theories emerged. The germ-line theories maintained that the genome contributed by the germ cells, egg and sperm, contains a large repertoire of immunoglobulin genes; thus, these theories invoked no special genetic mechanisms to account for antibody 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 contrast, the somatic-variation theories maintained that the genome contains a relatively small number of immunoglobulin genes, from which a large number of antibody specificities are generated in the somatic cells by mutation or recombination. As the amino acid sequences of more and more immunoglobulins were determined, it became clear that there must be mechanisms not only for generating antibody diversity 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 mechanism generated the variable (V) region? Neither the germ-line nor the somatic-variation proponents 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 diversify 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 variableregion sequences were associated with both and heavychain 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 associated with , , and heavy-chain constant regions. Considerable 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 immunoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant region). They suggested that these two genes must somehow come together at the DNA level to form a continuous message 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 somaticvariation theories) was that it could account for those immunoglobulins in which a single V region was combined with various C regions. By postulating a single constantregion 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 using 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, indirect evidence was not enough to overcome stubborn resistance 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 intellectual understanding of Ig-gene organization progressed ahead of the available methodology. Although the Dreyer and Bennett model provided a theoretical framework for reconciling the dilemma between Ig-sequence data and gene organization, actual validation of their hypothesis had to wait for several 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 endonucleases 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. Tonegawa and Hozumi suggested that, during differentiation of lymphocytes from the embryonic state to the fully differentiated 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 locus by comparing the fragments produced by digestion of DNA from a clone of B-lineage cells with the pattern obtained 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 rearranged 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 fragments generated by the treatment of DNA with a restriction enzyme is determined by the sequence of the DNA.The digestion of rearranged DNA with a restriction enzyme (RE) yields a pattern of restriction fragments that differ from those obtained by digestion of an unrearranged locus with the same RE. Typically, the fragments are analyzed by the technique of Southern blotting. In this example, a probe that includes a J gene segment is used to identify RE digestion fragments that include all or portions of this segment. As shown, rearrangement 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. Furthermore, rearranged DNA gives rise to novel fragments that are absent from digests of DNA in the germ-line configuration. This can be useful because both B cells and non-B cells have two immunoglobulin 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 locus, digestion of DNA from a myeloma or normal B-cell clone will produce a pattern of restriction that includes all of those in a germline 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 unrearranged 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 revealed than had been predicted by Dreyer and Bennett. The and light chains and the heavy chains are encoded by separate multigene families situated on different chromosomes (Table 5-1). In germ-line DNA, each of these multigene families contains several coding sequences, called gene segments, 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 segments; the rearranged VJ segments encode the variable region of the light chains. The heavy-chain family contains V, D, J, and C gene segments; the rearranged VDJ gene segments 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 molecule. 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 actually encoded by two gene segments appeared when Tonegawa 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 region corresponded to the nucleotide codon sequence, the remaining 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, encoded 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 unrearranged 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 defective 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 encode the variable region of the light chain, and each of the three functional C gene segments encodes the constant region 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 segments, the human lambda complex contains many V, J, and C pseudogenes. -CHAIN MULTIGENE FAMILY The -chain multigene family in the mouse contains approximately 85 V gene segments, each with an adjacent leader sequence 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 encode 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 humans, 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:
8536d_ch05_105-136 8/22/02 2: 46 PM Page 110 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immuno 110 PART I Generation of B-Cell and T-Cell Response VISUALIZING CONCEPTS (a)A-chain DNA L V22 2C2人4C4 廿[[}∥[日[}3 2.01.3 191.4 1.71.3 kbkb (b) K-chain DNA 5凵… (c)He 5 一-…HTH FICURE 5-3 Organization of immunoglobulin germ-line gene segments. The distances in kilobases(kb) separating the various segments in the mouse: (a)A light chain, (b)K light chain, and (c) gene segments in mouse germ-line DNA are shown below each avy chain. The A and K light chains are encoded by V, ), and c chain diagram gene segments. The heavy chain is encoded by V, D, ), and C gene was found to encode amino acids 98 to 113; however, neither sequence a short distance upstream. Downstream from the of these gene segments carried the information to encode DH gene segments are six functional JH gene segments, fol amino acids 95 to 97. When the nucleotide sequence was de- lowed by a series of CH gene segments. Each CH gene seg- termined for a rearranged myeloma DNA and compared ment encodes the constant region of an immunoglobulin with the germ-line DNA sequence, an additional nucleotide heavy-chain isotype. The Ch gene segments consist of coding sequence was observed between the VH and JH gene seg- exons and noncoding introns. Each exon encodes a separate ments. This nucleotide sequence corresponded to amino domain of the heavy-chain constant region. A similar heavy acids 95 to 97 of the heavy chain. chain gene organization is found in the mouse From these results, Hood and his colleagues proposed that The conservation of important biological effector func- third germ-line gene segment must join with the VHand h tions of the antibody molecule is maintained by the limited gene segments to encode the entire variable region of the number of heavy-chain constant-region genes. In humans eavy chain. This gene segment, which encoded amino acids and mice, the Ch gene segments are arranged sequentially in within the third complementarity-determining region the order Cu, Ca, Cy ce Ca( see Figure 5-3c). This sequential (CDR3), was designated D for diversity, because of its contri- arrangement is no accident; it is generally related to the se- bution to the generation of antibody diversity. Tonegawa and quential expression of the immunoglobulin classes in the his colleagues located the d gene segments within mouse course of B-cell development and the initial IgM response of germ-line DNA with a cDNA probe complementary to the d a B cell to its first encounter with an antigen. region, which hybridized with a stretch of DNA lying be tween the VH and JH gene segments The heavy-chain multigene family on human chromo- Variable-Region Gene ome 14 has been shown by direct sequencing of DNA to Rearrangements contain 51 VH gene segments located upstream from a clus- ter of 27 functional DH gene segments. As with the light- The preceding sections have shown that functional genes chain genes, each VH gene segment is preceded by a leader that encode immunoglobulin light and heavy chains are
was found to encode amino acids 98 to 113; however, neither of these gene segments carried the information to encode amino acids 95 to 97. When the nucleotide sequence was determined for a rearranged myeloma DNA and compared with the germ-line DNA sequence, an additional nucleotide sequence was observed between the VH and JH gene segments. This nucleotide sequence corresponded to amino acids 95 to 97 of the heavy chain. From these results, Hood and his colleagues proposed that a third germ-line gene segment must join with the VH and JH gene segments to encode the entire variable region of the heavy chain. This gene segment, which encoded amino acids within the third complementarity-determining region (CDR3), was designated D for diversity, because of its contribution to the generation of antibody diversity. Tonegawa and his colleagues located the D gene segments within mouse germ-line DNA with a cDNA probe complementary to the D region, which hybridized with a stretch of DNA lying between the VH and JH gene segments. The heavy-chain multigene family on human chromosome 14 has been shown by direct sequencing of DNA to contain 51 VH gene segments located upstream from a cluster of 27 functional DH gene segments. As with the lightchain genes, each VH gene segment is preceded by a leader sequence a short distance upstream. Downstream from the DH gene segments are six functional JH gene segments, followed by a series of CH gene segments. Each CH gene segment encodes the constant region of an immunoglobulin heavy-chain isotype. The CH gene segments consist of coding exons and noncoding introns. Each exon encodes a separate domain of the heavy-chain constant region. A similar heavychain gene organization is found in the mouse. The conservation of important biological effector functions of the antibody molecule is maintained by the limited number of heavy-chain constant-region genes. In humans and mice, the CH gene segments are arranged sequentially in the order C, C, C, C , C (see Figure 5-3c). This sequential arrangement is no accident; it is generally related to the sequential expression of the immunoglobulin classes in the course of B-cell development and the initial IgM response of a B cell to its first encounter with an antigen. Variable-Region Gene Rearrangements The preceding sections have shown that functional genes that encode immunoglobulin light and heavy chains are 110 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS 5′ 3′ 1.3 kb 1.7 kb 1.4 kb 19 kb 1.3 kb 2.0 kb 1.2 kb 70 kb Vλ2 Jλ2 Cλ2 Jλ4 Cλ4 Vλ1 Jλ3 Cλ3 J L L λ1 Cλ1 ψ (a) λ-chain DNA 5′ 3′ 2.5 kb 23 kb Vκn Jκ Cκ (b) κ-chain DNA n = ∼85 ψ L V Vκ1 L κ2 L 5′ 3′ 34 kb 55 kb 4.5 kb 6.5 kb VH1 Cµ Cγ3 (c) Heavy-chain DNA n = ∼134 VHn D DH13 H1 JH1 Cδ JH4 Cγ1 Cγ2b Cγ2a Cε Cα 21 kb 15 kb 14 kb 12 kb L L FIGURE 5-3 Organization of immunoglobulin germ-line gene segments in the mouse: (a) light chain, (b) light chain, and (c) heavy chain. The and light chains are encoded by V, J, and C gene segments. The heavy chain is encoded by V, D, J, and C gene segments. The distances in kilobases (kb) separating the various gene segments in mouse germ-line DNA are shown below each chain diagram. 8536d_ch05_105-136 8/22/02 2:46 PM Page 110 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
8536d_ch05_105-136 8/22/02 2: 46 PM Page 111 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 111 assembled by recombinational events at the DNa level. These Light-Chain DNA Undergoes vents and the parallel events involving T-receptor genes are V- Rearrangements the only known site-specific DNA rearrangements in verte brates. Variable-region gene rearrangements occur in an or- Expression of both k and A light chains requires rearrange dered sequence during B-cell maturation in the bone marrow. ment of the variable-region V and J gene segments In hu The heavy-chain variable-region genes rearrange first, then mans, any of the functional Va genes can combine with any the light-chain variable-region genes. At the end of this of the four functional Jx-Cx combinations In the mouse process, each B cell contains a single functional variable- things are slightly more complicated. DNA rearrangement region DNA sequence for its heavy chain and another for its can join the val gene segment with either the Jal or the J.3 gene segment, or the V,2 gene segment can be joined witl The process of variable-region gene rearrangement pro- the Ja2 gene segment. In human or mouse k light-chain duces mature, immunocompetent B cells; each such cell is DNA, any one of the Vk gene segments can be joined with committed to produce antibody with a binding site encoded any one of the functional J gene segments by the particular sequence of its rearranged V genes. As de- Rearranged K and A genes contain the following regions in scribed later in this chapter, rearrangements of the heavy- order from the 5 to 3 end: a short leader(L)exon, a non chain constant-region genes will generate further changes in coding sequence(intron), a joined V] gene segment, a second he immunoglobulin class(isotype)expressed by a B cell, but intron, and the constant region. Upstream from each leader hose changes will not affect the cells antigenic specificity. gene segment is a promoter sequence. The rearranged light- The steps in variable-region gene rearrangement occur in chain sequence is transcribed by RNA polymerase from the L an ordered sequence, but they are random events that result exon through the C segment to the stop signal, generating a in the random determination of B-cell specificity. The order, light-chain primary RNa transcript( Figure 5-4). The in mechanism, and consequences of these rearrangements are trons in the primary transcript are removed by RNA- described in this section processing enzymes, and the resulting light-chain messenge K-chain dna 5 HHHH v-J Jx Jx CK …LH adenylation RNA splicing mRNA L VJ CK Nascent polypeptide LVJ CK FIGURE 5.4 Kappa light-chain gene rearrangement and RNA pro- cessing events required to generate a k light-chain protein example, rearrangement joins V23 and J4
assembled by recombinational events at the DNA level. These events and the parallel events involving T-receptor genes are the only known site-specific DNA rearrangements in vertebrates. Variable-region gene rearrangements occur in an ordered sequence during B-cell maturation in the bone marrow. The heavy-chain variable-region genes rearrange first, then the light-chain variable-region genes. At the end of this process, each B cell contains a single functional variableregion DNA sequence for its heavy chain and another for its light chain. The process of variable-region gene rearrangement produces mature, immunocompetent B cells; each such cell is committed to produce antibody with a binding site encoded by the particular sequence of its rearranged V genes. As described later in this chapter, rearrangements of the heavychain constant-region genes will generate further changes in the immunoglobulin class (isotype) expressed by a B cell, but those changes will not affect the cell’s antigenic specificity. The steps in variable-region gene rearrangement occur in an ordered sequence, but they are random events that result in the random determination of B-cell specificity. The order, mechanism, and consequences of these rearrangements are described in this section. Light-Chain DNA Undergoes V-J Rearrangements Expression of both and light chains requires rearrangement of the variable-region V and J gene segments. In humans, any of the functional V genes can combine with any of the four functional J-C combinations. In the mouse, things are slightly more complicated. DNA rearrangement can join the V1 gene segment with either the J1 or the J3 gene segment, or the V2 gene segment can be joined with the J2 gene segment. In human or mouse light-chain DNA, any one of the V gene segments can be joined with any one of the functional J gene segments. Rearranged and genes contain the following regions in order from the 5 to 3 end: a short leader (L) exon, a noncoding sequence (intron), a joined VJ gene segment, a second intron, and the constant region. Upstream from each leader gene segment is a promoter sequence. The rearranged lightchain sequence is transcribed by RNA polymerase from the L exon through the C segment to the stop signal, generating a light-chain primary RNA transcript (Figure 5-4). The introns in the primary transcript are removed by RNAprocessing enzymes, and the resulting light-chain messenger Organization and Expression of Immunoglobulin Genes CHAPTER 5 111 FIGURE 5-4 Kappa light-chain gene rearrangement and RNA processing events required to generate a light-chain protein. In this example, rearrangement joins V23 and J4. Germ-line κ-chain DNA 5′ Vκ1 Vκ23 Vκn Jκ Cκ 3′ 3′ Vκ Jκ Vκ Jκ Jκ Jκ Cκ Rearranged κ-chain DNA V-J joining 3′ Cκ Transcription Primary RNA transcript mRNA VJ Cκ Nascent polypeptide VJ Cκ V J κ light chain Cκ Polyadenylation RNA splicing Translation Vκ Cκ (A)n Poly-A tail ψ L 5′ Vκ1 5′ L L L L L L L 8536d_ch05_105-136 8/22/02 2:46 PM Page 111 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
8536d_ch05_105-136 8/22/02 2: 47 PM Page 112 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 112 PART I Generation of B-Cell and T-Cell Response RNA then exits from the nucleus. The light-chain mRNa starting from the 5 end: a short L exon, an intron,a binds to ribosomes and is translated into the light-chain pro- VDJ segment, another intron, and a series of C gene seg tein. The leader sequence at the amino terminus pulls the ments. As with the light-chain genes, a promoter sequence is growing polypeptide chain into the lumen of the rough en- located a short distance upstream from each heavy-chain doplasmic reticulum and is then cleaved, so it is not present leader sequence in the finished light-chain protein product. Once heavy-chain gene rearrangement is accomplished, RNA polymerase can bind to the promoter sequence and Heavy- Chain DNA Undergoes transcribe the entire heavy-chain gene, including the introns V-D-/Rearrangements Initially, both Cu and Ca gene segments are transcribed Dif- ferential polyadenylation and RNA splicing remove the in Generation of a functional immunoglobulin heavy-chain trons and process the primary transcript to generate mRNA gene requires two separate rearrangement events within the including either the CH or the Cs transcript. These two variable region. As illustrated in Figure 5-5, a DH gene seg- mRNAs are then translated, and the leader peptide of the re- ment first joins to a JH segment; the resulting DHJH segment sulting nascent polypeptide is cleaved, generating finished p then moves next to and joins a VH segment to generate a and 8 chains. The production of two different heavy-chain VHDHH unit that encodes the entire variable region. In mRNAs allows a mature, immunocompetent B cell to express a rearranged gene consisting of the following sequences, surface. and lgD with identical antigenic specificity on its heavy-chain DNA, variable-region rearrangement produces both IgM Germ-lin H-chain 一高凸 Primary RNA transcript Polyadenylation LVDJ CH L VDJ C5 L V DJ L V DJ C5 u heavy chain 8 heavy chain FICURE 5-5 Heavy-chain gene rearrangement and RNA process. genes, although generally similar to expression of light-chain genes. ing events required to generate finished u or 8 heavy-chain protein. involves differential RNA processing, which generates several differ Two DNA joinings are necessary to generate a functional heavy-chain ent products, including u or 8 heavy chains. Each C gene is drawn as ene: a DH to JH joining and a VH to DHlh joining In this example, a single coding sequence; in reality, each is organized as a series of VH21, DH7, and JH3 are joined. Expression of functional heavy-chai ons and introns
RNA then exits from the nucleus. The light-chain mRNA binds to ribosomes and is translated into the light-chain protein. The leader sequence at the amino terminus pulls the growing polypeptide chain into the lumen of the rough endoplasmic reticulum and is then cleaved, so it is not present in the finished light-chain protein product. Heavy-Chain DNA Undergoes V-D-J Rearrangements Generation of a functional immunoglobulin heavy-chain gene requires two separate rearrangement events within the variable region. As illustrated in Figure 5-5, a DH gene segment first joins to a JH segment; the resulting DHJH segment then moves next to and joins a VH segment to generate a VHDHJH unit that encodes the entire variable region. In heavy-chain DNA, variable-region rearrangement produces a rearranged gene consisting of the following sequences, starting from the 5 end: a short L exon, an intron, a joined VDJ segment, another intron, and a series of C gene segments. As with the light-chain genes, a promoter sequence is located a short distance upstream from each heavy-chain leader sequence. Once heavy-chain gene rearrangement is accomplished, RNA polymerase can bind to the promoter sequence and transcribe the entire heavy-chain gene, including the introns. Initially, both C and C gene segments are transcribed. Differential polyadenylation and RNA splicing remove the introns and process the primary transcript to generate mRNA including either the C or the C transcript. These two mRNAs are then translated, and the leader peptide of the resulting nascent polypeptide is cleaved, generating finished and chains. The production of two different heavy-chain mRNAs allows a mature, immunocompetent B cell to express both IgM and IgD with identical antigenic specificity on its surface. 112 PART II Generation of B-Cell and T-Cell Responses FIGURE 5-5 Heavy-chain gene rearrangement and RNA processing events required to generate finished or heavy-chain protein. Two DNA joinings are necessary to generate a functional heavy-chain gene: a DH to JH joining and a VH to DHJH joining. In this example, VH21, DH7, and JH3 are joined. Expression of functional heavy-chain genes, although generally similar to expression of light-chain genes, involves differential RNA processing, which generates several different products, including or heavy chains. Each C gene is drawn as a single coding sequence; in reality, each is organized as a series of exons and introns. Primary RNA transcript mRNA Nascent polypeptide 5′ Germ-line VH1 VHn DH1 DH7 DH13 JH H-chain DNA D-J joining 5′ Rearranged VH1 VH20 J V DJ H H-chain DNA Transcription V J Polyadenylation RNA splicing D µ heavy chain V J D V J D L L L L L L L 3′ Cµ Cδ Cγ3 Cγ1 Cγ2b Cγ2a Cε Cα 3′ Cµ Cδ Cγ3 Cγ1 Cγ2b Cγ2a Cε Cα Cµ Translation (A)n Cµ Cµ V J D Cδ Translation (A)n V J D Cδ V J D Cδ or or or δ heavy chain 3′ V J D Cµ Cδ 5′ L L L 5′ 3′ VH1 C VH21 DH1 DH6 DH JH µ Cδ Cγ3 Cγ1 Cγ2b Cγ2a Cε Cα V-DJ joining L L L VHn 8536d_ch05_105-136 8/22/02 2:47 PM Page 112 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
8536d_ch05_105-136 8/22/02 2: 47 PM Page 113 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 113 spacer In heavy-chain DNA, the signal sequences of the VH Mechanism of Variable-Region and JH gene segments have two-turn spacers, the signals on DNA Rearrangements either side of the DH gene segment have one-turn spacers (Figure 5-6b) Signal sequences having a one-turn spacer can Now that we've seen the results of variable-region gene re- join only with sequences having a two-turn spacer(the so- arrangements, let's examine in detail how this process occurs called one-turn/two-turn joining rule). This joining rule en- during maturation of B cells. sures,for example, that a Vi segment joins only to a JL segment and not to another Vi segment; the rule likewise en Recombination Signal Sequences sures that VH, DH, and JH segments join in proper order and Direct recombination that segments of the same type do not join each other. The discovery of two closely related conserved sequences in Gene Segments Are Joined by Recombinases derstanding of the mechanism of gene rearrangements. DNA -(D)-)recombination, which takes place at the junctions sequencing studies revealed the presence of unique recombi- between RSSs and coding sequences, is catalyzed by enzymes nation signal sequences(RSSs)flanking each germ-line V, collectively called v(D) recombinase D, and J gene segment. One RSS is located 3 to each V gene Identification of the enzymes that catalyze recombination segment, 5,to each gene segment, and on both sides of each of V, D, and gene segments began in the late 1980s and is still D gene segment. These sequences function as signals for the ongoing. In 1990 David Schatz, Marjorie Oettinger, and recombination process that rearranges the genes. Each RSS David Baltimore first reported the identification of two contains a conserved palindromic heptamer and a conserved recombination-activating genes, designated RAG-Iand AT-rich nonamer sequence separated by an intervening se- RAG-2, whose encoded proteins act synergistically and are re- quence of 12 or 23 base pairs (Figure 5-6a). The intervening quired to mediate V-(D)-1joining. The RAG-1 and RAG-2 pro- 12-and 23-bp sequences correspond, respectively, to one and teins and the enzyme terminal deoxynucleotidyl transferase are the only lymphoid-specific gene products that called one-turn recombination signal sequences and two. have been shown to be involved in V-(D)-)rearrangement. The recombination of variable-region gene segments The Vk signal sequence has a one-turn spacer, and the k consists of the following steps, catalyzed by a system of re- gnal sequence has a two-turn spacer In A light-chain DNA, mbinase enzymes(Figure 5-7) this order is reversed; that is, the va signal sequence has a Recognition of recombination signal sequences(RSSs) wo-turn spacer, and the Jx signal sequence has a one-turn by recombinase enzymes, followed by synapsis in which (a) Nucleotide sequence of RSSs GTGTCAC-23 bpFTGTTTTTGG CCAAAAACA-12bpFGTGACAC Heptamer Nonamer Nonamer Heptamer Two-turn RSS One-turn rss b) Location of RSSs in germ-line immunoglobulin DNA K-chain DNA … h Heavv- chain DNA5′ …◆…“}3 FICURE5-6 Two conserved sequences in light-chain and heavy. RSS-designated one-turn RSS and two-turn RSS-have charac chain DNA function as recombination signal sequences(RSSs). teristic locations within A-chain, K-chain, and heavy-chain germ- (a)Both signal sequences consist of a conserved palindromic hep- line DNA During DNA rearrangement, gene segments adjacent to tamer and conserved AT-rich nonamer; these are separated by the one-turn RSS can join only with segments adjacent to the two- nonconserved spacers of 12 or 23 base pairs. (b) The two types of turn RSS
Mechanism of Variable-Region DNA Rearrangements Now that we’ve seen the results of variable-region gene rearrangements, let’s examine in detail how this process occurs during maturation of B cells. Recombination Signal Sequences Direct Recombination The discovery of two closely related conserved sequences in variable-region germ-line DNA paved the way to fuller understanding of the mechanism of gene rearrangements. DNA sequencing studies revealed the presence of unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment. One RSS is located 3 to each V gene segment, 5 to each J gene segment, and on both sides of each D gene segment. These sequences function as signals for the recombination process that rearranges the genes. Each RSS contains a conserved palindromic heptamer and a conserved AT-rich nonamer sequence separated by an intervening sequence of 12 or 23 base pairs (Figure 5-6a). The intervening 12- and 23-bp sequences correspond, respectively, to one and two turns of the DNA helix; for this reason the sequences are called one-turn recombination signal sequences and twoturn signal sequences. The V signal sequence has a one-turn spacer, and the J signal sequence has a two-turn spacer. In light-chain DNA, this order is reversed; that is, the V signal sequence has a two-turn spacer, and the J signal sequence has a one-turn spacer. In heavy-chain DNA, the signal sequences of the VH and JH gene segments have two-turn spacers, the signals on either side of the DH gene segment have one-turn spacers (Figure 5-6b). Signal sequences having a one-turn spacer can join only with sequences having a two-turn spacer (the socalled one-turn/two-turn joining rule). This joining rule ensures, for example, that a VL segment joins only to a JL segment and not to another VL segment; the rule likewise ensures that VH, DH, and JH segments join in proper order and that segments of the same type do not join each other. Gene Segments Are Joined by Recombinases V-(D)-J recombination, which takes place at the junctions between RSSs and coding sequences, is catalyzed by enzymes collectively called V(D)J recombinase. Identification of the enzymes that catalyze recombination of V, D, and J gene segments began in the late 1980s and is still ongoing. In 1990 David Schatz, Marjorie Oettinger, and David Baltimore first reported the identification of two recombination-activating genes, designated RAG-1 and RAG-2, whose encoded proteins act synergistically and are required to mediate V-(D)-J joining. The RAG-1 and RAG-2 proteins and the enzyme terminal deoxynucleotidyl transferase (TdT) are the only lymphoid-specific gene products that have been shown to be involved in V-(D)-J rearrangement. The recombination of variable-region gene segments consists of the following steps, catalyzed by a system of recombinase enzymes (Figure 5-7): ■ Recognition of recombination signal sequences (RSSs) by recombinase enzymes, followed by synapsis in which Organization and Expression of Immunoglobulin Genes CHAPTER 5 113 FIGURE 5-6 Two conserved sequences in light-chain and heavychain DNA function as recombination signal sequences (RSSs). (a) Both signal sequences consist of a conserved palindromic heptamer and conserved AT-rich nonamer; these are separated by nonconserved spacers of 12 or 23 base pairs. (b) The two types of RSS—designated one-turn RSS and two-turn RSS—have characteristic locations within -chain, -chain, and heavy-chain germline DNA. During DNA rearrangement, gene segments adjacent to the one-turn RSS can join only with segments adjacent to the twoturn RSS. (a) Nucleotide sequence of RSSs CACAGTG GTGTCAC 23 bp 23 bp ACAAAAACC TGTTTTTGG Heptamer Nonamer Two-turn RSS 12 bp 12 bp Nonamer One-turn RSS Heptamer CACTGTG GTGACAC GGTTTTTGT CCAAAAACA (b) Location of RSSs in germ-line immunoglobulin DNA 5′ 3′ VH Heavy-chain DNA DH JH CH 5′ 3′ Vκ κ-chain DNA Jκ Cκ 5′ 3′ Vλ λ-chain DNA J L λ Cλ L L 8536d_ch05_105-136 8/22/02 2:47 PM Page 113 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
8536d_ch05_105-136 8/22/02 2: 47 PM Page 114 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 114 PART I Generation of B-Cell and T-Cell Response (a) Deletional joining (b) Inversional joining two signal sequences and the adjacent coding sequences a A reaction catalyzed by RAg-1 and RAG-2 in which the by RAG-1/2 and synapsis free 3-OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence, simultaneously producing a hairpin structure at the cut end of the coding sequence and a flush, 5-phosphorylated, double-strand break at the by RAG-1/2 Cutting of the hairpin to generate sites for the addition leonides, followed by the 3 few nucleotides from the coding sequence by a single- strand endonuclease ③ Hairpin formation a Addition of up to 15 nucleotides, called N-region and double- strand nucleotides, at the cut ends of the V, D, and J coding DNA break by sequences of the heavy chain by the enzyme terminal RAG- deoxynucleotidyl transferase Repair and ligation to join the coding sequences and to join the signal sequences, catalyzed by normal double- 和mmk strand break repair(DSBr)enzymes Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, be of p-nucleotides 非 tween the RSSs. The transcriptional orientation of the gene segments to be joined determines the fate of the signal joint and intervening DNA. When the two gene segments are in the same transcriptional orientation, joining results in dele tion of the signal joint and intervening DNA as a circular ex cision product(Figure 5-8). Less frequently, the two gene of N-nucleotides by tdT segments have opposite orientations. In this case joining oc- curs by inversion of the DNA, resulting in the retention of Coding joint to form joints by Gnetum rss Two-turn RSS FIGURES-7 Model depicting the general process of recombina tion of immunoglobulin gene segments is illustrated with Vx and Jk (a)Deletional joining occurs when the gene segments to be joined have the same transcriptional orientation(indicated by horizontal blue arrows). This process yields two products: a rearranged v) unit that includes the coding joint, and a circular excision product con- sisting of the recombination signal sequences(RSSs), signal joint, and intervening DNA. (b) Inversional joining occurs when the gene FIGURE 5-8 Circular DNA isolated from thymocytes in which the segments have opposite transcriptional orientations. In this case, the DNA encoding the chains of the T-cell receptor(TCR)undergoes re- RSSS, signal joint, and intervening DNA are retained, and the orien- arrangement in a process like that involving the immunoglobulin tation of one of the joined segments is inverted. In both types of re- genes. Isolation of this circular excision product is direct evidence for combination, a few nucleotides may be deleted from or added to the the mechanism of deletional joining shown in Figure 5-7.[From K. ends of the coding sequences before they are rejoin Okazaki et al., 1987, Cell 49: 477 J
two signal sequences and the adjacent coding sequences (gene segments) are brought into proximity ■ Cleavage of one strand of DNA by RAG-1 and RAG-2 at the junctures of the signal sequences and coding sequences ■ A reaction catalyzed by RAG-1 and RAG-2 in which the free 3-OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence, simultaneously producing a hairpin structure at the cut end of the coding sequence and a flush, 5-phosphorylated, double-strand break at the signal sequence ■ Cutting of the hairpin to generate sites for the addition of P-region nucleotides, followed by the trimming of a few nucleotides from the coding sequence by a singlestrand endonuclease ■ Addition of up to 15 nucleotides, called N-region nucleotides, at the cut ends of the V, D, and J coding sequences of the heavy chain by the enzyme terminal deoxynucleotidyl transferase ■ Repair and ligation to join the coding sequences and to join the signal sequences, catalyzed by normal doublestrand break repair (DSBR) enzymes Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, between the RSSs. The transcriptional orientation of the gene segments to be joined determines the fate of the signal joint and intervening DNA. When the two gene segments are in the same transcriptional orientation, joining results in deletion of the signal joint and intervening DNA as a circular excision product (Figure 5-8). Less frequently, the two gene segments have opposite orientations. In this case joining occurs by inversion of the DNA, resulting in the retention of 114 PART II Generation of B-Cell and T-Cell Responses (a) Deletional joining 5′ 3′ Vκ Jκ RSS 5′ 3′ Vκ Jκ (b) Inversional joining 3′ 5′ 3′ Recognition of RSSs by RAG-1/2 and synapsis L J Vκ κ Coding joint 5′ 3′ Signal joint Signal joint Coding joint Single-strand DNA cleavage by RAG-1/2 Hairpin formation and double-strand DNA break by RAG-1/2 Random cleavage of hairpin by endonuclease generates sites for the addition of P-nucleotides Optional addition to H-chain segments of N-nucleotides by TdT Repair and ligation of coding and signal sequences to form joints by DSBR enzymes 1 2 3 4 5 = Two-turn RSS = One-turn RSS L L + FIGURE 5-7 Model depicting the general process of recombination of immunoglobulin gene segments is illustrated with V and J. (a) Deletional joining occurs when the gene segments to be joined have the same transcriptional orientation (indicated by horizontal blue arrows). This process yields two products: a rearranged VJ unit that includes the coding joint, and a circular excision product consisting of the recombination signal sequences (RSSs), signal joint, and intervening DNA. (b) Inversional joining occurs when the gene segments have opposite transcriptional orientations. In this case, the RSSs, signal joint, and intervening DNA are retained, and the orientation of one of the joined segments is inverted. In both types of recombination, a few nucleotides may be deleted from or added to the cut ends of the coding sequences before they are rejoined. FIGURE 5-8 Circular DNA isolated from thymocytes in which the DNA encoding the chains of the T-cell receptor (TCR) undergoes rearrangement in a process like that involving the immunoglobulin genes. Isolation of this circular excision product is direct evidence for the mechanism of deletional joining shown in Figure 5-7. [From K. Okazaki et al., 1987, Cell 49:477.] 8536d_ch05_105-136 8/22/02 2:47 PM Page 114 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
