Class switch recombination of the chicken IgH chain genes: implications for the primordial switch region repeats

Hiroyuki Kitao1,2,3, Hiroshi Arakawa1,2,7, Kei-ichi Kuma1, Hideo Yamagishi1,4, Naoto Nakamura5, Shuichi Furusawa5, Haruo Matsuda5, Masahiro Yasuda6, Shigeo Ekino6 and Akira Shimizu2

1 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
2 Center for Molecular Biology and Genetics, Kyoto University, Kyoto 606-8507, Japan
3 Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan
4 Health Research Foundation, Kyoto 606-8225, Japan
5 Department of Immunobiology, Faculty of Applied Biological Science, Hiroshima University, Hiroshima 739-8528, Japan
6 Department of Anatomy, Kumamoto University Medical School, Kumamoto 860-0811, Japan

Correspondence to: A. Shimizu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In mammals and the amphibian, Xenopus, isotypes of antibodies have been shown to be changed through class switch recombination within the IgH chain gene locus. Here, we identified switch (S) repetitive sequences in the 5' introns of the Ig Cµ and C{gamma} genes of the chicken. The Sµ region is composed of two homologous regions, Sµ1 and Sµ2. The Sµ1 region is an upstream 3.7 kb sequence composed of 37 repeats of a consensus sequence containing tandem repeats of the decamer ACCAGTATGG. The Sµ2 region is a downstream 1.4 kb sequence consisting of simple tandem repeats of a decamer CCCAGTACAG. The S{gamma} region contains repeats of the decamer TATGGGGCAG. Analysis of chicken IgG-producing hybridomas revealed that the Cµ gene was deleted from the chromosome by the recombination occurring between the Sµ and S{gamma} regions. Recombination breakpoints at the Cµ gene of splenocytes from an immunized chicken were scattered around the Sµ region and two such breakpoints, the precise position of which were determined, were located within possible hairpin loop structures at the palindromic sequence of Sµ1. A primordial palindromic sequence from which the prevalent switch repeat motifs of mammals, chickens and amphibians may have diverged is presented.

Keywords: chicken switch region, deletional recombination, palindrome, prevalent pentamer, repetitive sequence


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During the development of B lymphocytes in mammals, the IgH chain gene undergoes two types of DNA recombination. One is V(D)J recombination, which assembles segments of variable region exons (1). The generation of a complete V(D)J sequence upstream of the Cµ gene induces the expression of the Cµ gene. The µ H chain is the first IgH class expressed during B cell ontogeny. As B cells differentiate, other classes of IgH molecules are produced, following the second type of rearrangement, class switch recombination, that is the main molecular mechanism of IgH class or isotype switching. During class switching, the V(D)J structure and antigen binding specificity remain unchanged (24). IgH class switching occurs via deletion of constant region (CH) genes between a V exon and the CH gene to be expressed (4,5). Structural characterization of rearranged CH genes has shown that switch recombination takes place in switch (S) regions composed of tandem repetitive sequences which are located 5' of each CH gene, except for C{delta} (69). Analysis of a large number of switch junction sequences has revealed that recombination occurs between non-homologous sequences in broad but defined areas including or surrounding the S regions (10,11). Two molecular mechanisms of DNA rearrangement, the intrachromosomal recombination model (5,12) and the unequal sister chromatid exchange model (13), have been proposed for mammalian class switch recombination. The intrachromosomal recombination model was supported by the detection of excised switch circles of various sizes (1416).

IgH isotypes related to those in mammals first appeared in amphibians (17,18). Xenopus possesses three isotypes, i.e. IgM, IgY and IgX (19), which belong to the same linkage group. IgY is an IgG-equivalent isotype and is expressed after IgM, implying a IgM to IgY class switch takes place (20). IgX behaves as a functional analog of IgA (21) and IgX expression is shown as the result of a switch event (19). Indeed, the existence of S regions in Xenopus µ and {chi} genes was shown, and the switch junctions of deletional recombination were identified (22). However, Xenopus antibodies mature only slightly in affinity during immune responses in spite of significant somatic point mutations, possibly from the lack of an effective selection mechanism in the absence of germinal centers. The dominant Ig isotype in the serum of Xenopus is IgM, probably due to the lack of memory and/or lower efficiency of class switching.

The Ig diversification mechanism of chicken B cell is unique among vertebrates. There is only one functional V segment each for the H and L chain. The V region sequence is diversified after V(D)J recombination by gene conversion using the pseudogene sequence located upstream to the functional V segment (23,24). Gene conversion occurs not only in the bursa of Fabricius but also in splenic germinal centers (25). The chicken has three constant region isotypes, i.e. IgM, IgG (or IgY) and IgA (2628), and IgG is the major isotype in the adult serum. However, precise organization of the chicken CH genes including their order on the chromosome is not known yet. IgM to IgG class switching occurs in splenic germinal centers during T cell-dependent immune responses (29).

We previously found a region homologous to mouse Sµ and S{alpha} regions containing tandem repeats of pentamers (C/T) (C/A)CAG in the 5' intron of the Cµ exon of the chicken IgH gene locus (30). These results suggest a similar recombination mechanism in both chicken and mammalian IgH class switching. On the other hand, the chicken has evolved a unique mechanism for the diversification of the V region sequence using gene conversion, and there might be a common process in the molecular mechanism of somatic hypermutation, gene conversion and class switch recombination since these processes are shown to be dependent on transcription (4,31,32). In mammals, models for the expression of IgH isotypes other than µ, such as alternative splicing of a long primary transcript producing VH–C{delta} mRNA (33) and trans-splicing between germline C{gamma} transcripts and VH–Cµ transcripts yielding VH–C{gamma} mRNA, are also proposed (3436). Therefore, chicken IgH class switching might be executed by a unique mechanism other than switch recombination.

In this work, the chicken S regions of µ and {gamma} genes were analyzed. The switch junctions generated by deletional recombination mediated by these Sµ and S{gamma} regions were identified. The sequences of S regions and switch junctions between Sµ and S{gamma} led us to suggest the recombinogenic nature of the S regions is conserved in mammals, chickens and the amphibian Xenopus.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chicken strain and cells
White Leghorn, near-inbred line H-B 15 (formerly V) chickens were housed in Kumamoto University, Japan (37). Spleen cells were isolated from an 11-week-old female chicken i.v. immunized 7 days earlier with (4-hydroxy-3-iodo-5-nitrophenyl)acetyl coupled to BSA. Liver cells and red blood cells were isolated from unimmunized chickens of the similar age.

IgG-producing chicken hybridomas
Two chicken hybridomas, HUC2-13 (38), which produces IgG antibody against mammalian prion protein, and HUC1-2, and their parental chicken B cell line MuH1 (39) were used in this study. These cell lines were maintained in RPMI 1640 medium containing 10% FCS at 42°C under 5% CO2. The HUC1-2 hybridoma was obtained from the same fusion experiment which established the HUC2-13 hybridoma and produces IgG of unknown specificity (unpublished data).

Genomic Southern hybridization
Genomic DNA (10 µg) was digested with HindIII or PstI, loaded on 0.5% agarose gel electrophoresis and hybridized with the probes shown in Fig. 1Go. Nucleotide sequences of the probes obtained by PCR were confirmed after subcloning.



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Fig. 1. Partial restriction map of the chicken IgH Cµ and C{gamma} gene locus (A), deletion fragments (ag and AE) used for sequencing (B) and clones containing the Sµ/S{gamma} switch junction (C). Arrowheads show the orientation of oligonucleotide primers; uSµ1 (with EcoRI site), 5'-ACGAATTCGCACTAATTAGCGGGG-3'; uCµ1-2 (with BamHI site), 5'-ACGGATCCGGAAACGGAACTGTTGTTG-3'; uC{gamma}1-6 (with XhoI site), 5'-ACCCTCGAGTCGGAACAACAGGCGG-3'; F, M13 universal forward primer; R, M13 universal reverse primer; EA-1, positions 399–414 of database AB029075; EA-2, positions 920–939; EA-R1, positions 2496–2515; and EA-R2, positions 1780–1800. Bars above the map show the probes; DJ-6-1, a 2.7 kb ApaI and EcoRI fragment of DJ-6 (23); M1, a 1.1 kb SpeI fragment of pE13-A (30); Cµ-U, a 285 bp PmaCI and SacII fragment of the Igµ chain cDNA prepared from the chicken B cell line DT40 (accession no. X01613, positions 132–416) (30); C{gamma}-U, a 180 bp PCR fragment of the CH1 exon of the C{gamma} gene, obtained from H-B15 chicken genomic DNA by PCR (accession no. X07174, positions 270–449) (27); and G1, a 270 bp Sau3AI and XhoI fragment of the 5' flanking region of the chicken C{gamma} gene (accession no. AB029078). Deletion fragment lengths are 2713 (a), 2209 (b), 1946 (c), 1714 (d), 1264 (e), 814 (f), 482 (g), 1533 (A), 1471 (B), 1211 (C), 729 (D) and 295 (E) bp. The 8050 (AB029075) and 611 (AB029077) bp sequences we analyzed and locations of Sµ1, Sµ2 and S{gamma} are bracketed. Clones 1-2C and 2-8A are PCR-amplified fragments with uSµ1 and uC{gamma}1-6 primers from spleen cells. A, ApaI; E, EcoRI; H, HindIII; K, KpnI; Pm, PmaCI; Sa, SacII; Sm, SmaI; Sp, SpeI; X, XbaI.

 
Genomic PCR amplification
Oligonucleotide primers for amplification of the fragments of the IgH gene locus were designed as shown in Fig. 1Go(A). Template DNA (150 ng) was subjected to PCR amplification in a 50 µl reaction mixture containing 2.5 U DNA polymerase, KOD-DASH (Toyobo, Osaka, Japan). PCR amplification was performed in 30 cycles consisting of 20 s at 94°C, 30 s at 58°C and 3 min at 72°C. Specifically amplified fragments were detected by using M1 and G1 probes (Fig. 1Go).

Cloning and sequencing
A genomic library containing partial Sau3AI digests of liver DNA of local stock White Leghorn chicken in {lambda}EMBL-3 was screened with the C{gamma}-U probe. One positive clone (c{lambda}G-1) was obtained. PCR of the phage DNA of c{lambda}G-1 using uC{gamma}1-6 and EMBL3 (5'-ACTCGTGAAAGGTAGGC-3') primers was performed to isolate the 5' flanking region of the chicken C{gamma} gene yielding a 270 bp fragment. All genomic fragments and PCR-amplified fragments were subcloned into pBluescript KS (+) (Stratagene, La Jolla, CA) (30). Deletional clones from the pE13-A clone containing the JH–Cµ intron of H-B 15 chicken (30) were sequenced (Fig. 1BGo). SURE competent cells (Stratagene) were used when PCR-amplified fragments were subcloned to minimize cloning artifacts. All clones were sequenced by using the Dye Terminator cycle sequencing kit with AmpliTaq DNA polymerase, FS (Applied Biosystems, Foster City, CA), and analyzed using a model 373A DNA sequencer (Applied Biosystems).

Homology plot and sequence alignment
Homology plot comparison between identical pairs of S region sequences was carried out by a computer program based on the method described previously (40). Gaps were introduced by manual operation to obtain the optimal alignment of nucleotide sequences among internal repeats of the S region.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Repetitive sequences in the 5' intron of chicken Cµ and C{gamma} genes
We previously detected a region that cross-hybridizes with mouse Sµ and S{alpha} probes within the chicken IgH gene locus. This region contains tandem repeats of pentamer (C/T) (C/A)CAG (30). We further determined the 8050 bp nucleotide sequence of the JH–Cµ intron as shown in Fig. 1Go (EMBL/Genbank/DDBJ database accession no. AB029075). The sequence we previously reported (30) is located at positions 6518–7010 of this 8050 bp. Homology matrix analysis revealed three clusters of repeats within this 8050 bp sequence (Fig. 2AGo). Two of them, located downstream (position 2725–7852) are continuous and homologous to each other. The most 5' repetitive region (position 1155–2220), consisting of the most AT-rich repeats of pentamers, ATTAG and AAATA, has no sequence homology with the downstream repetitive sequences (position 2725–7852). Since the downstream repetitive sequences have homology with mouse Sµ and S{alpha} regions (30), we view this repeat as defining the borders of the Sµ region. The chicken Sµ region is composed of two homologous regions, Sµ1 and Sµ2 (Fig. 2AGo). A search for repetitions of G nucleotides revealed that the Sµ1 region is composed of 37 repeats of DNA segments whose lengths are 70–112 bp (102 bp consensus sequence) (Fig. 3AGo). Each segment contains tandem repeats (3–8 times) of the decamer ACCAGTATGG. Eight nucleotides of this decamer are well conserved (Fig. 3AGo) though the eighth T and ninth G occasionally change to C and A respectively. This tandem repeat is occasionally interrupted by another decamer GTGCACTGGG, which includes an ApaLI recognition site. Two or three repeats of a pentamer CTGGT, complementary to ACCAG of the prevalent decamer (Fig. 3AGo), are found between repetitions of G nucleotides and tandem repeats of the prevalent decamer ACCAGTATGG. At the boundaries of these two pentamers, KpnI sites are often present. In contrast, the Sµ2 region consists of simple tandem repeats of the decamer CCCAGTACAG and its slight variants. Among these 10 nucleotides, 4 nucleotides (second C, third C, fourth A and sixth T) are more conserved (Fig. 3 BGo) and 99% of the repeats have these consensus bases.



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Fig. 2. Internal homologies of the chicken Sµ and S{gamma} regions. (A) Homology matrix between identical Sµ 8050 bp sequence (accession no. AB029075). Locations of Sµ1 and Sµ2 regions are bracketed below the matrix, and the recombination breakpoints of clones 1-2C and 2-8A are shown by arrowheads. (B) Homology matrix between identical S{gamma} 611 bp sequence (accession no. AB029077). Location of the S{gamma} region is bracketed below the matrix. Segments of 20 bp having >80% homology to other segments are shown.

 



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Fig. 3. Alignment of nucleotide sequences in Sµ1 (A), Sµ2 (B) and S{gamma} (C) regions. The numbers correspond to the position in database accession nos AB029075 (A and B) and AB029077 (C). Only bases that differ from the consensus sequence are shown and hyphens denote identical bases. Prevalent decamers of the consensus sequence are underlined and the frequency (%) of occurrence of the consensus bases at each position is shown below the decamers. Inverted repeats of prevalent pentamers ACCAG and CTGGT are indicated by a pair of superscript divergent arrows, and the recombination breakpoints in clones 1-2C and 2-8A are shown by triangles in (A). Y, pyrimidine; K, G or T.

 
We were unable to obtain a germline genomic clone containing a long 5' flanking sequence of the chicken C{gamma} gene probably due to instability in bacteria and/or a biased distribution of restriction sites caused by the highly repetitious sequence. Therefore we used another strategy to obtain a longer partial sequence of the chicken S{gamma} region. We obtained two clones (1-2C and 2-8A) from PCR products amplified from the genomic DNA of spleen cells from an immunized chicken with uSµ1 and uC{gamma}1-6 primers (Figs 1 and 5GoGo) carrying the Sµ/S{gamma} rearranged structure (see below for details). The 611 bp sequence at the 3' end (sequenced from the uC{gamma}1-6 primer) of these two clones was identical except for 4 base changes (EMBL/Genbank/DDBJ database accession no. AB029077) indicating that this sequence is from the 3' part of the S{gamma} region and 5' part of the C{gamma} gene which is the same as the germline sequence. When the 611 bp sequence is run against itself using dot matrix analysis, the 3' border of S{gamma} is seen graphically as the end of the repetitive sequence (Fig. 2BGo). This repetitive sequence is composed of repeats of DNA segments whose lengths are 22–60 bp (40 bp consensus sequence) (Fig. 3CGo). Each segment contains at least one prevalent decamer TATGGGGCAG and, occasionally, its variant TATGGGTCTC was duplicated. Seven nucleotides in this prevalent decamer are well conserved (Fig. 3CGo) with the seventh G, ninth A and 10th G occasionally changing to T, T and C respectively.



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Fig. 5. PCR amplification of Sµ (A and C) and Sµ/S{gamma} rearranged (B and D) fragments. (A and B) PCR products amplified with uSµ1/uCµ1-2 (A) and uSµ1/uC{gamma}1-6 (B) from cell lines HUC2-13 (lanes 1 and 4), HUC1-2 (lanes 2 and 5) and MuH1 (lanes 3 and 6). DNA was separated on agarose gel and hybridized with M1 and G1 as indicated. (C and D) PCR products amplified with uSµ1/uCµ1-2 (C) and uSµ1/uC{gamma}1-6 (D) from spleen (C, lanes 1; D, lanes 1–3 and 6–8), liver (C, lane 2; D, lanes 4 and 9) and red blood cells (C, lanes 3; D, lanes 5 and 10) were hybridized with M1 and G1 probes as shown in (A and B). HindIII digests of {lambda}DNA were used as size markers.

 
Cµ gene deletion in IgG-producing chicken hybridomas
Repetitive sequences in the 5' intron of Cµ and C{gamma} genes suggest that class switch recombination is mediated by these S repetitive sequences. In mammals and Xenopus, class switch recombination is accompanied by deletion of the intervening C genes from the chromosome (5,22). To investigate whether chicken IgG-producing cells delete the intervening Cµ gene from the chromosome, the IgH gene loci of chicken hybridomas, HUC1-2 and HUC2-13, which produce IgG antibodies to mammalian prion protein (38), were analyzed by genomic Southern hybridization using DJ-6-1, Cµ-U and C{gamma}-U probes (Fig. 4Go). When genomic DNA of these hybridomas was digested with HindIII, both C{gamma}-U and DJ-6-1 probes detected additional 22 (HUC2-13) and 26 (HUC1-2) kb fragments, differing from three bands (>35, 23 and 13 kb) of their parental cell line MuH1 (Fig. 4A–CGo). The results suggest that the HindIII fragments detected only in these hybridomas include the recombinant sequence that hybridizes with C{gamma}-U and DJ-6-1 probes. The recombinant HindIII fragments differed by 4 kb (26–22 kb) in length between the two hybridomas. HindIII digestion analysis was consistent with PstI digestion analysis. Both DJ-6-1 and C{gamma}-U probes detected additional 13 (HUC2-13) and 17 (HUC1-2) kb fragments, differing from the three bands (30, 22 and 8.5 kb) detected in the MuH1 (Fig. 4D–FGo). The common PstI fragments again differed by 4 kb (17–13 kb) in length between the two hybridomas. Cµ-U probe detected identical germline bands and no additional bands in both hybridomas and MuH1 (Fig. 4C and FGo). Collectively, these results suggest that Cµ genes in the IgG-producing allele of the two hybridomas were deleted and had not translocated to other regions of the genome.



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Fig. 4. Hybridization of IgG-producing hybridomas, HUC2-13 (lane 1) and HUC1-2 (lane 2), and their parent cell line MuH1 (lane 3) DNA with the probes C{gamma}-U (A and D), DJ-6-1 (B and E), Cµ-U (C), and a mixture of Cµ-U and C{alpha}-U (F). DNA was cut with HindIII (A–C) and PstI (D–F). C{alpha}-U is a 189 bp PCR fragment of the CH1 exon of C{alpha} (positions 430–618 of database accession no. S40610) and the hybridized bands are shown by asterisks. Molecular sizes of the bands were calculated with HindIII or XhoI digests of {lambda}DNA as markers.

 
Sµ/S{gamma} recombination within the genome of IgG-producing chicken cells
To confirm that these hybridomas and immunized chicken splenocytes contained the IgH gene locus exhibiting rearrangement between Sµ and S{gamma} regions, we carried out PCR amplification of recombinants with uSµ1 and uC{gamma}1-6 primers (Fig. 5Go). A 5.5 kb fragment was amplified with uSµ1 and uCµ1-2 primers from DNA from each cell line and tissue examined (Fig. 5A and CGo). Amplification with uSµ1 and uC{gamma}1-6 primers under the same conditions gave the common 2.0 (HUC2-13) and 5.8 (HUC1-2) kb fragments hybridizing with both M1 and G1 probes (Fig. 5BGo, lanes 1, 2 and 4, 5). No PCR fragment hybridizing with both probes was amplified from MuH1 DNA (Fig. 5BGo, lanes 3 and 6) although some bands were detected only by the G1 probe probably due to PCR artifacts being seen. Such bands of PCR artifacts hybridizing with only one probe, especially with the G1 probe, were also seen in the amplification from the hybridoma DNA (Fig. 5BGo, lanes 1, 2 and 4, 5). The PCR fragments of the recombined DNA differed by 3.8 kb (5.8–2.0 kb) in length between the two hybridomas. Thus, the recombinants identified both by restriction enzyme digestion and PCR showed a consistent size difference between these two hybridomas. As they were not amplified from MuH1 DNA (Fig. 5BGo, lanes 3 and 6), rearranged bands (5.8 and 2.0 kb) are derived from the genome of IgG-producing splenocytes that had fused with MuH1.

Similar PCR amplification was performed using DNA from splenocytes, liver and red blood cells of H-B 15 chicken. The uSµ1 and uC{gamma}1-6 primers amplified several fragments from genomic DNA of splenocytes from an in vivo immunized chicken which hybridized with both M1 and G1 probes (Fig. 5DGo, lanes 1–3 and 6–8). Such fragments hybridized with both M1 and G1 probes were not amplified from genomic DNA isolated from liver cells or red blood cells (Fig. 5DGo, lanes 4, 5 and 9, 10) though G1 probe again detected some PCR artifacts (Fig. 5DGo, lanes 9 and 10). In contrast to IgG-producing hybridomas, the fragments amplified from spleen genomic DNA were different from one reaction to the other even when we used the same batch of genomic DNA (Fig. 5DGo, lanes 1–3 and 6–8). This difference using the same DNA means the exact target molecules were different in each reaction, as amplification using the hybridoma (monoclonal) DNA repeatedly gave the same bands (data not shown). In our experimental conditions, it is estimated that each PCR reaction contained DNA from a few hundred IgG+ B cells. Our result indicates that random pick up of a few hundred IgG+ cells from total spleen gave different combinations of clones. Since both Sµ and S{gamma} regions are tandem repeats of unit sequences, the relative intensities of the hybridized bands detected by each probe may differ, reflecting the relative length of each region in the amplified fragments as shown in Fig. 5Go(D). These results indicate that IgG-producing cells in spleen are polyclonal and that switch recombination in splenic B cells occurred at various positions within the S regions.

Identification of Sµ/S{gamma} recombination sites
To identify the sites of Sµ/S{gamma} recombination, we size-fractionated the fragments amplified from spleen cells into two fractions containing 9.4–4.0 or 4.5–1.3 kb fragments and cloned two fragments (1-2C and 2-8A) from each fraction (Fig. 1CGo). The inserts of clones 1-2C and 2-8A were 4.1 and 3.1 kb respectively. These clones contained expected Sµ and C{gamma} gene sequences at each end (data not shown). Compared with the germline Sµ sequence, these clones contained no gross deletion in the 5' starting 545 bp Sµ sequence except for one base change at position 3006 in clone 1-2C. To determine the nucleotide sequence of Sµ/S{gamma} recombination junctions in these two clones, we prepared subclones using the nearest KpnI site in the germline Sµ1 sequence. Nucleotide sequencing allowed us to assign the recombination junctions to positions 5045 (1-2C) and 3841 (2-8A) within the germline Sµ region (Figs 2, 3 and 6GoGoGo). The 3' sequences of these junctions consist of repeats of the prevalent decamer specified in the S{gamma} sequence in the 5' intron of C{gamma} gene, TATGGGGCAG (Fig. 6Go). Clone 2-8A showed two single base changes at positions 3831 and 3833 in the Sµ1 sequence (Fig. 6BGo).



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Fig. 6. Nucleotide sequences surrounding Sµ/S{gamma} breakpoints of clones 1-2C (A) and 2-8A (B). The recombinant sequence is compared with the corresponding germline Sµ sequence registered in the database AB029075 and identical bases are shown by vertical lines. Prevalent decamer sequence motifs in the S{gamma} and Sµ1 are bracketed above and below the sequence respectively. Inverted repeats of prevalent pentamers, ACCAG and CTGGT, are indicated by a pair of superscript divergent arrows and the recombination breakpoints are shown by triangles. Sequence data of clones 1-2C and 2-8A are available from the EMBL/GenBank/DDBJ database under accession nos AB029079 and AB029080 respectively.

 

    Discussion
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 Abstract
 Introduction
 Methods
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 Discussion
 References
 
In this work, the complete and partial sequences of the Sµ and S{gamma} regions respectively, which are crucial for IgH class switching and structurally interesting, of a near-inbred line (H-B 15) of White Leghorn chicken were determined. These regions were first defined as S regions by the location of internal homology (Fig. 2Go), and homology with mouse Sµ and S{alpha} regions (30). The Cµ gene was deleted from genomic DNA of two IgG-producing hybridomas (Fig. 4C and FGo). This deletion should have occurred by the rearrangement between these Sµ and S{gamma} regions (Fig. 5BGo). The junction between the Sµ1 and S{gamma} regions was identified on the PCR-amplified fragment from the genome of the HUC2-13 hybridoma (sequence data are not shown but available from the EMBL/GenBank/DDBJ database under accession no. AB029081). We identified the sequences of two switch recombination junctions between the Sµ1 and S{gamma} regions of PCR-amplified fragments from spleen cells isolated from an immunized chicken (Fig. 6Go). PCR products of Sµ2/S{gamma} recombinants were also identified by Southern hybridization (data not shown). Thus, the S regions defined by the sequence actually include the regions responsible for class switch recombination.

We found two homologous repetitive sequences of internal homology, Sµ1 and Sµ2, in the Sµ region. Sµ1 is a tandem repetitive sequence of a 102 bp repeating unit consensus sequence containing a cluster of G nucleotides, two to three duplications of CTGGT, three to eight duplications of the decamer ACCAGTATGG, and occasional insertion of GTGCACTGGG. Prevalent pentamers ACCAG and TATGG constitute 27 and 19% of the Sµ1 region respectively. A pair of ACCAG and CTGGT duplications is a palindrome that may possibly form loop structures, preferred sites of enzymatic cleavage (4143). Sµ2 was made up of simple tandem repeats of the consensus decamer unit, CCCAGTACAG. Frequencies of prevalent pentamers CCCAG and TACAG are 21 and 23% respectively. S{gamma} was composed of tandem repeats of a consensus 40 bp unit that contains duplications of the decamer TATGGGGCAG and occasional duplications of TATGGGTCTC. The most prevalent pentamer TATGG appears at a frequency of 24% and GGCAG appears at 15%. These prevalent pentamers in the Sµ and S{gamma} regions may be derived from the primordial palindromic pentamer CTCAG after several rounds of mutation and duplication as suggested in Fig. 7Go.



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Fig. 7. Evolutionary relationship among prevalent pentamers in human, mouse, chicken and Xenopus S regions. The incidence (%) of prevalent pentamers in each S region is shown in parentheses. Possible base changes are underlined. The sequence data were obtained from the EMBL/GenBank/DDBJ database under accession nos X59795 for human Sµ (44), X59797 for human S{varepsilon} (44), J00442 for mouse Sµ (6), D11468 for mouse S{alpha} (45) and AF002166 for Xenopus Sµ (22).

 
Mammalian S regions tend to be rich in GC bases; 54.6–64.5% GC (6,44,45), as summarized in Table 1Go. In contrast, the Xenopus S regions are AT rich; 35.1% GC for Sµ (22). The chicken S regions are GC rich; 53.4–55.2% GC (Table 1Go). The average GC content of total DNA is 40.3% for human and mouse, 40.9% for Xenopus, and 45.0% for the chicken (46). Thus, the GC contents in the S regions may have deviated from the genomic GC content after rounds of segmental duplication. In spite of the different base compositions, the repetitive character and the palindromes appear to be conserved in the S region between mammals, birds and amphibians (22). In particular, a regular repeat containing pentameric key motif sequences extending over kilobases is a common feature of the chicken and most of mammalian S regions, although the key motifs are not identical between them. In this sense, the S region repeats of Xenopus are a bit different. They are less regular and the key motif seems to be a tetramer. These differences might be reflected in the difference of switching efficiency. Indeed, the switching to S{varepsilon}, the least regular repeat among mammalian S regions, seems to be less efficient.


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Table 1. GC content of various S regions
 
The sequence of the two PCR products amplified with specific primers of Sµ and S{gamma} showed that DNA breaks occurred within the Sµ and S{gamma} regions, followed by joining of the two S region sequences. The breakpoints in the Sµ region were assigned to positions 3841 (2-8A) and 5045 (1-2C) of the 8050 bp sequence (AB029075) located within the hairpin loop structures predicted from the palindromic sequences (Figs 3A and 6GoGo). Switch recombination does not seem to utilize the homology between the common pentamers, TATGG, prevalent in the Sµ1 and S{gamma} regions (Fig. 6Go). In human and Xenopus, switch junctions were more frequent at microsites corresponding to the transition from a stem to a loop structure formed by single-stranded DNA (22). The Ku70/Ku80 subunits bind to DNA double-strand breaks, nicks, gaps and hairpins in a sequence-independent manner (47). These structures also activate DNA-dependent protein kinase (DNA-PK) (48). Since every component of DNA-PK (catalytic subunit and the Ku70/Ku80 heterodimer) is essential for switch recombination (4951), Ku subunits Ku70 and Ku80 may be involved in the recognition of switch repetitive structures.

Switch repetitive sequences are susceptible to cleavage, producing DNA double-strand breaks at the neck region of the hairpin loop structure (42). Rejoining of DNA double-strand breaks by mammalian ligases in vitro are enhanced by the Ku complex (52). DNA double-strand breaks during switch recombination might be held together by Ku in a synaptic complex to enhance rejoining by ligases. The two breakpoints of class switch recombination identified in the chicken are consistent with the model of Ku-mediated non-homologous end joining (Fig. 6Go). Rejoining sites of two S regions are thought to be processed as misannealing sites by mismatch repair-dependent machinery (5355). The two possible point mutations found in the Sµ1 region flanking the switch junction of clone 2-8A (Fig. 6BGo) may be due to error-prone DNA synthesis, as suggested in the mouse process (10,56,57), although errors in PCR cannot be formally excluded.

A primordial palindromic pentamer, CTCAG, was deduced from the prevalent pentamers in the chicken S regions although this pentamer per se does not appear. As shown in Fig. 7Go, the primordial palindromic pentamer, CTGAG, was also deduced from the pentamers prevalent in human and mouse S regions. CTGAG is also the most prevalent pentamer in pig Sµ region (58). Xenopus Sµ region includes the palindromic tetramer AGCT motif. From these unique oligomeric repeats in mammals, chicken and Xenopus, we can deduce the ancestral primordial switch repeats, AGCT(G/C)AGCT (Fig. 7Go), although it is not the only one possible. Mammalian Sµ regions are mainly composed of the nonameric sequence, AGCTGAGCT (6,44,58). In the Xenopus Sµ region, the nonameric sequence AGCTCAGCT appears at a frequency much higher than the estimation of random appearance (12 times in 4182 bp Sµ sequence) (22). The recurrence of various oligomers from this palindromic, nonameric sequence might generate the repetitive occurrence of short palindromes essential for creating recombinogenic DNA structure.

As tandem repetitive sequences in general can arise independently and overall similarity in nucleotide sequence of S regions is not as high as the recombination signal sequences for V(D)J recombination, repetitive and palindromic organization alone does not assure the single origin of all the S regions. However, deletional recombination between repetitive sequences during class switching was shown to be the same between mammals, chicken and amphibians, and the prevalent repeat motifs of S regions led us to deduce the ancestral palindromic nonamer motif of the switch repeats. These results strongly support a single origin of S regions, and that the fine molecular mechanisms for the class switch recombination appear to be conserved during evolution except for those toward C{delta} which might be the oldest non-µ isotype (59) and have different mechanisms for switching (60). The fine molecular mechanisms of this recombination are still elusive, but this common ancestral palindrome might be the key to this mechanism conserved throughout evolution.


    Acknowledgments
 
We thank N. Takahashi for providing the liver genomic library of local stock White Leghorn chicken, M. Ikenaga for encouraging advice, M. Nazarea-Umemura and S. Fraser for critical reading of this manuscript, and Y. Hirano for technical assistance. This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan.


    Abbreviations
 
DNA-PK DNA-dependent protein kinase
S switch

    Notes
 
7 Present address: Department of Cellular Immunology, Heinrich-Pette-Institute, 20251 Hamburg, Germany Back

Transmitting editor: D. Kitamura

Received 11 January 2000, accepted 3 March 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
  2. Shimizu, A. and Honjo, T. 1984. Immunoglobulin class switching. Cell 36:801.[ISI][Medline]
  3. Esser, C. and Radbruch, A. 1990. Immunoglobulin class switching: molecular and cellular analysis. Annu. Rev. Immunol. 8:717.[ISI][Medline]
  4. Stavnezer, J. 1996. Antibody class switching. Adv. Immunol. 61:79.[ISI][Medline]
  5. Honjo, T. and Kataoka, T. 1978. Organization of immunoglobulin heavy chain genes and allelic deletion model. Proc. Natl Acad. Sci. USA 75:2140.[Abstract]
  6. Nikaido, T., Nakai, S. and Honjo, T. 1981. Switch region of immunoglobulin Cµ gene is composed of simple tandem repetitive sequences. Nature 292:845.[ISI][Medline]
  7. Kataoka, T., Miyata, T. and Honjo, T. 1981. Repetitive sequence in class-switch recombination regions of immunoglobulin heavy chain genes. Cell 23:357.[ISI][Medline]
  8. Nikaido, T., Yamawaki-Kataoka, Y. and Honjo, T. 1982. Nucleotide sequences of switch regions of immunoglobulin C{varepsilon} and C{gamma} genes and their comparison. J. Biol. Chem. 257:7322.[Abstract/Free Full Text]
  9. Shimizu, A., Takahashi, N., Yaoita, Y. and Honjo, T. 1982. Organization of the constant-region gene family of the mouse immunoglobulin heavy chain. Cell 28:499.[ISI][Medline]
  10. Dunnick, W., Hertz, G. Z., Scappino, L. and Gritzmacher, C. 1993. DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21:365.[Abstract]
  11. Lee, C. G., Kondo, S. and Honjo, T. 1998. Frequent but biased class switch recombination in the Sµ flanking regions. Curr. Biol. 8:227.[ISI][Medline]
  12. Jäck, H. M., McDowell, M., Steinberg, C. M. and Wabl, M. 1988. Looping out and deletion mechanism for the immunoglobulin heavy-chain class switch. Proc. Natl Acad. Sci. USA 85:1581.[Abstract]
  13. Obäta, M., Kataoka, T., Nakai, S., Yamagishi, H., Takahashi, N., Yamawaki-Kataoka, Y., Nikaido, T., Shimizu, A. and Honjo, T. 1981. Structure of a rearranged {gamma}1 chain gene and its implication to immunoglobulin class-switch mechanism. Proc. Natl Acad. Sci. USA 78:2437.[Abstract]
  14. von Schwedler, U., Jäck, H. M. and Wabl, M. 1990. Circular DNA is a product of the immunoglobulin class switch rearrangement. Nature 345:452.[ISI][Medline]
  15. Iwasato, T., Shimizu, A., Honjo, T. and Yamagishi, H. 1990. Circular DNA is excised by immunoglobulin class switch recombination. Cell 62:143.[ISI][Medline]
  16. Matsuoka, M., Yoshida, K., Maeda, T., Usuda, S. and Sakano, H. 1990. Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell 62:135.[ISI][Medline]
  17. Fellar, J. S., Kertourn, F., Wiles, M. V., Schwager, J. and Charlemagne, J. 1993. Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum {upsilon} chain deduced from cDNA sequence. Immunogenetics 38:311.[ISI][Medline]
  18. Mußmann, R., Wilson, M., Marcuz, A., Courtet, M. and Du Pasquier, L. 1996. Membrane exon sequences of the three Xenopus Ig classes explain the evolutionary origin of mammalian isotypes. Eur. J. Immunol. 26:409.[ISI][Medline]
  19. Hsu, E., Flajnik, M. F. and Du Pasquier, L. 1985. A third immunoglobulin class in amphibians. J. Immunol. 135:1998.[Abstract/Free Full Text]
  20. Hsu, E. and Du Pasquier, L. 1984. Ontogeny of the immune system in Xenopus II. Antibody repertoire differences between larvae and adults. Differentiation 28:116.[ISI]
  21. Mußmann, R., Du Pasquier, L. and Hsu, E. 1996. Is Xenopus IgX an analog of IgA? Eur. J. Immunol. 26:2823.[ISI][Medline]
  22. Mußmann, R., Courtet, M., Schwager, J. and Du Pasquier, L. 1997. Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals. Eur. J. Immunol. 27:2610.[ISI][Medline]
  23. Reynaud, C. A., Dahan, A., Anquez, V. and Weill, J. C. 1989. Somatic hyperconversion diversifies the single VH gene of the chicken with a high incidence in the D region. Cell 59:171.[ISI][Medline]
  24. Reynaud, C. A., Anquez, V., Grimal, H. and Weill, J. C. 1987. A hyperconversion mechanism generates the chicken light chain pre-immune repertoire. Cell 48:379.[ISI][Medline]
  25. Arakawa, H., Furusawa, S., Ekino, S. and Yamagishi, H. 1996. Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers. EMBO J. 15:2540.[Abstract]
  26. Dahan, A., Reynaud, C. A. and Weill, J. C. 1983. Nucleotide sequence of the constant region of a chicken µ heavy chain immunoglobulin mRNA. Nucleic Acids Res. 11:5381.[Abstract]
  27. Parvari, R., Avivi, A., Lentner, F., Ziv, E., Ter-Or, S., Burstein, Y. and Schechter, I. 1988. Chicken immunoglobulin {gamma}-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus. EMBO J. 7:739.[Abstract]
  28. Mansikka, A. 1992. Chicken IgA H chains. Implications concerning the evolution of H chain genes. J. Immunol. 149:855.[Abstract/Free Full Text]
  29. Jeurissen, S. H. M. 1993. The role of various compartments in the chicken spleen during are antigen-specific humoral response. Immunology 80:29.[ISI][Medline]
  30. Kitao, H., Arakawa, H., Yamagishi, H. and Shimizu, A. 1996. Chicken immunoglobulin µ-chain gene: germline organization and tandem repeats characteristic of class switch recombination. Immunol. Lett. 52:99.[ISI][Medline]
  31. Thompson, C. B. 1992. Creation of immunoglobulin diversity by intrachromosomal gene conversion. Trends. Genet. 8:416.[ISI][Medline]
  32. Diaz, M. and Flajnik, M. F. 1998. Evolution of somatic hypermutation and gene conversion in adaptive immunity. Immunol. Rev. 162:13.[ISI][Medline]
  33. Maki, R., Roeder. W., Traunecker, A., Sidman, C., Wabl, M., Raschke, W. and Tonegawa, S. 1981. The role of DNA rearrangement and alternative RNA processing in the expression of immunoglobulin delta genes. Cell 24:353.[ISI][Medline]
  34. Shimizu, A., Nussenzweig, M. C., Mizuta, T. R., Leder, P. and Honjo, T. 1989. Immunoglobulin double-isotype expression by trans-mRNA in a human immunoglobulin transgenic mouse. Proc. Natl Acad. Sci. USA 86:8020.[Abstract]
  35. Sideras, P., Mizuta, T. R., Kanamori, H., Suzuki, N., Okamoto, M., Kuze, K., Ohno, H., Doi, S., Fukuhara, S., Hassan, M. S., Hammarstrom, L., Smith, E., Shimizu, A. and Honjo, T. 1989. Production of sterile transcripts of C{gamma} genes in an IgM-producing human neoplastic B cell line that switches to IgG-producing cells. Int. Immunol. 1:631.[Medline]
  36. Shimizu, A., Nussenzweig, M. C., Han, H., Sanchez, M. and Honjo, T. 1991. Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene. J. Exp. Med. 173:1385.[Abstract]
  37. Kondo, T., Arakawa, H., Kitao, H., Hirota, Y. and Yamagishi, H. 1993. Signal joint of immunoglobulin V{lambda}1–J{lambda} and novel joints of chimeric V pseudogenes on extrachromosomal circular DNA from chicken bursa. Eur. J. Immunol. 23:245.[ISI][Medline]
  38. Matsuda H., Mitsuda, H., Nakamura, N., Furusawa, S., Mohri, S. and Kitamoto, T. 1999. A chicken monoclonal antibody with specificity for the N-terminal of human prion protein. FEMS Immunol. Med. Microbiol. 23:189.[ISI][Medline]
  39. Nishinaka, S., Akiba, H., Nakamura, M., Suzuki, K., Suzuki, T., Tsubokura, K., Horiuchi, H., Furusawa, S. and Matsuda, H. 1996. Two chicken B cell lines resistant to ouabain for the production of chicken monoclonal antibodies. J. Vet. Med. Sci. 58:1053.[ISI][Medline]
  40. Toh, H., Hayashida, H. and Miyata, T. 1983. Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. Nature 305:827.[ISI][Medline]
  41. Baar, J., Pennell, N. M. and Shulman, M. J. 1996. Analysis of a hotspot for DNA insertion suggests a mechanism for Ig switch recombination. J. Immunol. 157:3430.[Abstract]
  42. Wuerffel, R. A., Du, J., Thompson, R. J. and Kenter, A. L. 1997. Immunoglobulin S{gamma}3 DNA specific double stranded breaks are induced in mitogen activated B cells and are implicated in switch recombination. J. Immunol. 159:4139.[Abstract]
  43. Paull, T. T. and Gellert, M. 1998. The 3' to 5' exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks. Mol. Cell 1:969.[ISI][Medline]
  44. Mills, F. C., Brooker, J. S. and Camerini-Otero, R. D. 1990. Sequences of human immunoglobulin switch regions: implications for recombination and transcription. Nucleic Acids Res. 18:7305.[Abstract]
  45. Arakawa, H., Iwasato, T., Hayashida, H., Shimizu, A., Honjo, T. and Yamagishi, H. 1993. The complete murine immunoglobulin class switch region of the {alpha} heavy chain gene—hierarchic repetitive structure and recombination breakpoints. J. Biol. Chem. 268:4651.[Abstract/Free Full Text]
  46. Brown, T. A. 1991. Molecular Biology LABFAX. BIOS/Blackwell Scientific Publications, Oxford.
  47. Falzon, M., Fewell, J. W. and Kuff, E. L. 1993. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J. Biol. Chem. 268:10546.[Abstract/Free Full Text]
  48. Morozov, V. E., Falzon, M., Anderson, C. W. and Kuff, E. L. 1994. DNA-dependent protein kinase is activated by nicks and large single-stranded gaps. J. Biol. Chem. 269:16684.[Abstract/Free Full Text]
  49. Rolink, A., Melchers, F. and Andersson, J. 1996. The SCID but not the RAG-2 gene product is required for Sµ-S{varepsilon} heavy chain class switching. Immunity 5:319.[ISI][Medline]
  50. Manis, J. P., Gu, Y., Lansford, R., Sonoda, E., Ferrini, R., Davidson, L., Rajewsky, K. and Alt, F. W. 1998. Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187:2081.[Abstract/Free Full Text]
  51. Casellas, R., Nussenzweig, A., Wuerffel, R., Pelanda, R., Reichlin, A., Suh, H., Qin, X. F., Besmer, E., Kenter, A., Rajewsky, K. and Nussenzweig, M. C. 1998. Ku80 is required for immunoglobulin isotype switching. EMBO J. 17:2404.[Abstract/Free Full Text]
  52. Ramsden, D. A. and Gellert, M. 1998. Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks. EMBO J. 17:609.[Abstract/Free Full Text]
  53. Kenter, A. L. 1999. The liaison of isotype class switch and mismatch repair: an illegitimate affair. J. Exp. Med. 190:307.[Free Full Text]
  54. Ehrenstein, M. R. and Neuberger, M. S. 1999. Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. EMBO J. 18:3484.[Abstract/Free Full Text]
  55. Schrader, C. E., Edelmann, W., Kucherlapati, R. and Stavnezer, J. 1999. Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J. Exp. Med. 190:323.[Abstract/Free Full Text]
  56. Dunnick, W., Wilson, M. and Stavnezer, J. 1989. Mutations, duplications, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch. Mol. Cell. Biol. 9:1850.[ISI][Medline]
  57. Du, J., Zu, Y., Shanmugam, A. and Kenter, A. L. 1997. Analysis of immunoglobulin S{gamma}3 recombination breakpoints by PCR: implications for the mechanism of isotype switching. Nucleic Acids Res. 25:3066.[Abstract/Free Full Text]
  58. Sun, J. and Butler, J. E. 1997. Sequence analysis of pig switch µ, Cµ, and Cµm. Immunogenetics 46:452.[ISI][Medline]
  59. Wilson, M., Bengtén, E., Miller, N. W., Clem, L. W., Du Pasquier, L. and Warr, G. W. 1997. A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc. Natl Acad. Sci. USA 94:4593.[Abstract/Free Full Text]
  60. Kluin, P. M., Kayano, H., Zani, V. J., Kluin-Nelemans, H., C., Tucker, P. W., Satterwhite, E. and Dyer, M. J. S. 1995. IgD class switching: identification of a novel recombination site in neoplasmic and normal B cells. Eur. J. Immunol. 25:3504.[ISI][Medline]