《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 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 de￾termined 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 seg￾ments. 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 contri￾bution 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 be￾tween the VH and JH gene segments. The heavy-chain multigene family on human chromo￾some 14 has been shown by direct sequencing of DNA to contain 51 VH gene segments located upstream from a clus￾ter of 27 functional DH gene segments. As with the light￾chain 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, fol￾lowed by a series of CH gene segments. Each CH gene seg￾ment 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 heavy￾chain gene organization is found in the mouse. The conservation of important biological effector func￾tions 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 se￾quential 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 verte￾brates. Variable-region gene rearrangements occur in an or￾dered 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 variable￾region DNA sequence for its heavy chain and another for its light chain. The process of variable-region gene rearrangement pro￾duces 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 de￾scribed later in this chapter, rearrangements of the heavy￾chain 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 rearrange￾ment of the variable-region V and J gene segments. In hu￾mans, 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 non￾coding 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 light￾chain 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 in￾trons in the primary transcript are removed by RNA￾processing 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 pro￾cessing 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 pro￾tein. The leader sequence at the amino terminus pulls the growing polypeptide chain into the lumen of the rough en￾doplasmic 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 seg￾ment 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 seg￾ments. 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. Dif￾ferential polyadenylation and RNA splicing remove the in￾trons 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 re￾sulting 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 process￾ing 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 differ￾ent 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 re￾arrangements, 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 un￾derstanding of the mechanism of gene rearrangements. DNA sequencing studies revealed the presence of unique recombi￾nation 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 se￾quence 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 two￾turn 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 so￾called one-turn/two-turn joining rule). This joining rule en￾sures, for example, that a VL segment joins only to a JL segment and not to another VL segment; the rule likewise en￾sures 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 re￾quired to mediate V-(D)-J joining. The RAG-1 and RAG-2 pro￾teins 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 re￾combinase 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 heavy￾chain DNA function as recombination signal sequences (RSSs). (a) Both signal sequences consist of a conserved palindromic hep￾tamer 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 charac￾teristic locations within -chain, -chain, and heavy-chain germ￾line DNA. During DNA rearrangement, gene segments adjacent to the one-turn RSS can join only with segments adjacent to the two￾turn 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 single￾strand 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 double￾strand break repair (DSBR) enzymes Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, be￾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 segments have opposite orientations. In this case joining oc￾curs 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 recombina￾tion 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 con￾sisting 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 orien￾tation of one of the joined segments is inverted. In both types of re￾combination, 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 re￾arrangement 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: