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Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters
Molecular Immunology 38 (2001) 557–564
Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters Tereza Ventura-Holman, Craig J. Lobb∗ Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA Received 1 July 2001; accepted 10 August 2001
Abstract Two structurally-related genomic clusters of catfish immunoglobulin heavy chain gene segments are known. The first gene cluster contains DH and JH segments, as well as the C region exons encoding the functional C. The second gene cluster contains multiple VH gene segments representing different VH families, a germline-joined VDJ, a single JH segment, and at least two pseudogene C exons. It was not known whether these gene clusters were linked, nor was the organization or the location of VH segments associated within the first gene cluster known. Pulsed-field gel electrophoresis studies have been used to determine the structural organization of these gene clusters. Restriction mapping studies show that the two gene clusters are closely linked; the second gene cluster is located upstream from the first with the C regions within the clusters separated by about 725 kb. The clusters are in the same relative transcriptional orientation, and the results indicate that the complete IgH locus spans no more than 1000 kb and may be as small as 750–800 kb. VH gene segments are located both upstream and downstream of the pseudo-C exons; however, no VH gene segments that hybridized with the VH specific probes were detected downstream of the functional C. These studies coupled with earlier sequence analyses indicate that the catfish IgH locus arose from a massive internal duplication event. Subsequent gene rearrangement within the duplicated cluster likely resulted in the presence of the germline VDJ and the deletion of intervening V, D and J segments. Transposition by a member of the Tc1/mariner family of transposable elements appears to have led to the disruption of the duplicated C. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Antibodies; Molecular biology; Comparative immunology; Gene evolution
1. Introduction Although the general structure of immunoglobulin V, D, J and C region segments has been conserved during vertebrate phylogeny, the evolution of the genomic organization and utilization patterns of these gene segments has been a dynamic process. The most familiar form of genomic organization is that represented in humans and mice, wherein various functional VH segments representing different VH families are located upstream from DH segments that are, in turn, located upstream of JH and C region segments (Tonegawa, 1983; Brodeur et al., 1988; Yancopoulos and Alt, 1986; Matsuda et al., 1998). A second organizational pattern is represented in chickens. The locus resembles that of humans and mice in that there are multiple VH segments upstream of DH, JH, and CH segments; however, in this case, only a single VH segment is functional. The other VH segments are pseudogenes, and their contribution to the diversity of the Ig Abbreviations: H, heavy chain of immunoglobulin; L, light chain of immunoglobulin; Mb, megabase; PFGE, pulsed-field gel electrophoresis ∗ Tel.: +1-601-984-1700; fax: +1-601-984-1708. E-mail address: [email protected] (C.J. Lobb).
repertoire is manifested only through gene conversion mechanisms (Reynaud et al., 1987; McCormack et al., 1991). A third organizational pattern is that represented in sharks, where there are multiple clusters of Ig gene segments. Within each cluster there are V, D, J, and C segments, and gene rearrangement appears restricted to the segments within individual clusters (Kokubu et al., 1988; Schluter et al., 1997). Recent studies have shown that the organization of Ig genes in channel catfish reflects a phylogenetic “anthology” of different genomic organizational patterns of segmental Ig genes. The L chains, represented by two different classes, are both organized in multiple gene clusters wherein V, J and C gene segments reside within each cluster. The VL segments are in inverted transcriptional orientation relative to the J and C segments, which indicates that VL segments likely undergo rearrangement to JL–CL segments by inversion (Ghaffari and Lobb, 1993; Ghaffari and Lobb, 1997). In contrast, the H chain locus contains only a single functional C gene that encodes the four domain C region of the predominant serum antibody and Ig (Ghaffari and Lobb, 1989a,b; Lobb, 1985). The JH locus contains nine functional JH segments that are tightly clustered within a 2.2 kb region located immediately upstream from C (Hayman
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et al., 1993). The DH locus was recently identified through the examination of circular recombination excision products which proved that germline D–J recombination can result in deletion events. This locus contains at least three different DH segments that represent single member gene families, and is located about 8.6 kb upstream of the JH locus (Hayman and Lobb, 2000). In addition, it has been shown that VH gene families extensively diverged at the level of the bony fish. Southern blot studies indicate an extensive genomic VH repertoire consisting of at least seven VH gene families which represent >100 different gene segments (reviewed in Ventura-Holman et al., 1996). Examination of lambda genomic libraries indicate that members of the different gene families are interspersed with one another and that VH segments are closely linked; the average distance between segments is about 3.5 kb (Ventura-Holman et al., 1994). These results indicate that the structure, genomic organization, and recombination patterns of H chain genes that are typically associated with higher vertebrates evolved early in phylogeny at the level of the bony fish. Recently, a second cluster of channel catfish H chain gene segments was defined (Ghaffari and Lobb, 1999). The characterized 25 kb region of this second H chain cluster consists of multiple VH segments, representing different VH families located upstream of a germline-joined VDJ segment. The VDJ has a split leader sequence and a single open reading frame consistent with that known in expressed members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene C exons, C1 and C2. These exons are multiply crippled with RNA splice sites destroyed, and open reading frames interrupted by termination codons, insertions, and/or deletions. A 10.8 kb sequenced region was readily aligned with homologous JH and C exons in the first gene cluster, which indicated that this second cluster of H chain gene segments shared a close structural relationship with gene segments in the first cluster. With these results it was essential to characterize the genomic organization of these two H chain gene clusters. This report characterizes this relationship and provides new insights into the mechanisms underlying early evolutionary patterns of the IgH locus.
2. Materials and methods 2.1. Preparation and restriction of genomic DNA in agarose blocks for analysis by pulsed-field gel electrophoresis Erythrocytes from individual adult channel catfish (Ictalurus punctatus) were used as the source of genomic DNA for PFGE analyses. The genomic DNA agarose embedding procedure used in these studies is based on the procedure described by Smith et al. (1993), and modified as described in detail (Ventura-Holman and Lobb, 1995). Prior to restriction, agarose blocks containing 2–3 g of DNA were equili-
brated in three 15 min washes of the appropriate restriction buffer, and then incubated overnight with a maximum of 10 units of restriction enzyme/g DNA in 150 l of buffer at the recommended temperature (New England Biolabs, Beverly, MA). 2.2. PFGE running conditions: Southern blots, and hybridization conditions Molecular weight standards, prepared in agarose blocks by the manufacturer (Bio-Rad, Hercules, CA), were run in lanes adjacent to the restricted genomic DNA samples. The standards used in these experiments included: a lambda ladder for DNA fragment sizes ranging from 50 kb to 1 Mb; Saccharomyces cerevisiae chromosomes for sizes ranging from 200 kb to 2.2 Mb; and Hansenula wingei chromosomes for sizes ranging from 1 to 3.1 Mb. The molecular weight standards or restricted DNA was separated by agarose gel electrophoresis using a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad) using running conditions as described in the figure legends. For DNA fragments greater than 1 Mb in size, 0.8% agarose gels were prepared using 1X TAE in chromosomal grade agarose (Bio-Rad). For fragments less than 1 Mb, 1% gels in 0.5X TBE were prepared using pulsed-field certified agarose (Bio-Rad). The running buffer was the same as used to prepare the gel. The temperature of the running buffer was maintained at 14◦ C during electrophoresis. Following electrophoresis, the DNA was transferred to Zeta-Probe GT membranes (Bio-Rad) by capillary transfer, and cross-linked by UV irradiation as recommended by the manufacturer. The membranes were pre-washed in 3X SSC and 0.1% SDS at 65 ◦ C for 1 h and pre-hybridized for 6 h to overnight in 3X SSC, 5X Denhardt’s solution, 50% formamide and 0.1% SDS at 42 ◦ C. The blots were hybridized with radiolabeled probes in 3X SSC, 1X Denhardt’s solution, 50% formamide, and 0.2% SDS at 42 ◦ C overnight. The membranes were washed twice in 2X SSC, 0.1% SDS and four times in 0.2X SSC, 0.1% SDS at 65 ◦ C before autoradiography at −70 ◦ C (typically a 3-day exposure). For sequential hybridizations, the membranes were stripped of the radiolabeled probe by two 15 min washes in 0.5% SDS, 0.5X SSC. The boiling solution was poured over the blots and incubated at 85 ◦ C with shaking. Probes used in these studies included the following VH family-specific probes derived from cDNA clones previously described (Ghaffari and Lobb, 1991; Ventura-Holman et al., 1994; Ventura-Holman et al., 1996): a 300 bp PstI fragment from NG70 (VH1); a 378 bp PvuII fragment from NG41 (VH2); a 266 bp PstI fragment from NG54 (VH3); a 260 bp PvuII–BstEII fragment from NG10 (VH4); a 364 bp PvuII–BamHI fragment from NG66 (VH5); a 457 bp fragment from clone 10.4 (VH6); a 221 bp SstI–AvaI fragment from clone vh5e (VH7). The following probes were also used: a 778 bp EcoRI cDNA restriction fragment (containing the C3 and C4 domains of the functional C); a
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1.5 kb PstI–SstI restriction fragment of genomic clone C2, corresponding to a region (intron) located between the pseudogene C1 and C2 exons of the second H chain cluster (Ghaffari and Lobb, 1999); a 1.2 kb ClaI restriction fragment located within the cluster of JH segments found upstream of the functional C (designated as the JH probe, Hayman et al., 1993); and a 5.6 kb EcoRI restriction fragment located upstream of the JH locus in the first gene cluster (designated as the 5 -JH probe, Hayman and Lobb, 2000). Probes were radiolabeled with [32 P]dCTP to a specific activity of approximately 108 –109 cpm/g of DNA using either a multiprime or megaprime random priming kit (Amersham, Arlington Heights, IL).
3. Results 3.1. Linkage of the first and second H chain clusters Studies were initially undertaken to determine if the two catfish H chain gene clusters were linked on the same large genomic restriction fragments. The approach that was taken was to derive specific probes from C regions within each gene cluster and hybridize these probes to genomic restriction fragments that had been separated by PFGE. Agarose-embedded genomic DNA was restricted with either AscI or NotI, and following PFGE, the blot was hybridized with the cluster-specific C probes. These results showed that both probes hybridized with the same sized genomic fragment, an AscI fragment of 4.0 Mb or a NotI fragment of 5.7 Mb. The blots were sequentially hybridized with probes specific for each of the seven VH families, and in each case this identical-sized fragment was the only band that hybridized with these probes (data not shown). The linkage distance of the H chain gene clusters was narrowed with the use of additional 6 bp recognition site restriction enzymes. Genomic Southern blots of DNA restricted with NruI or EagI and hybridized with the cluster-specific C probes yielded single fragments of 1.55 or 1.45 Mb, respectively. When the blots were sequentially hybridized with specific probes for each of the seven VH families, these
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identical-sized fragments were the only fragments that were detected (Fig. 1). Double digests of genomic DNA restricted with both NruI and EagI revealed only the 1.45 Mb fragment when blots were hybridized with these probes. These combined analyses indicate that the two gene clusters are linked within 1.45 Mb and that all VH segments detected by these family-specific probes are contained on this size of fragment. 3.2. Linkage distance between the first and second H chain clusters The organization of the two H chain gene clusters within the 1.45 Mb region was further defined with the enzyme SfiI. The approach that was taken was to use restriction conditions that yielded partial digests of genomic DNA within the 100–1000 kb range. Southern blots of the PFGE-separated fragments were then sequentially hybridized with the cluster-specific C probes, and the restriction map of the overlapping fragments was determined. Fig. 2 shows the hybridization patterns of SfiI restricted DNA obtained from five individual outbred channel catfish. Three of these fish (fishes 9, 11 and 7, lanes 1, 3, and 5, respectively) yielded essentially the same hybridization patterns with the cluster-specific probes. Genomic DNA from the other two fish (fishes 10 and 12, lanes 2 and 4, respectively) yielded restriction patterns that were distinct from the other fish as well as somewhat different from each other. Representative restriction maps of these overlapping fragments obtained from these SfiI digests from these fish are shown in Fig. 3. These analyses showed that the SfiI fragment that contained the pseudo-C was closely linked to the fragment that contained that functional C. Two internal SfiI fragments separated the SfiI fragments defined by these cluster-specific C probes. The combined size of these two fragments ranged in size from 150–195 kb. The SfiI fragment that contained the functional C in fishes 9, 10, and 12 was about 310 or about 290 kb in fishes 11 and 7, respectively. In each case, this fragment was flanked downstream by a 240 kb SfiI fragment. However, the size of the SfiI fragments that hybridized with the specific probes for the pseudo C exhibited a size
Fig. 1. Southern blots of genomic catfish DNA (fish 4) restricted with EagI (lane 1) or NruI (lane 2) and separated by PFGE. The probes used for hybridization were specific for the C located within the first H chain gene cluster (panel A) or probes specific for the pseudo-C located within the second H chain gene cluster (panel B). Probes specific for each of the VH families 1–7 were sequentially hybridized with the blot and the results were identical. Representative results using the probe for the VH3 family are shown (panel C). The relative sizes of the fragments are shown in Mb units on the left of the panels. The pulsed-field electrophoresis parameters were: 0.8% agarose gel; 1X TAE; 2 V/cm; 106◦ included angle; initial pulse time 2 min; final pulse time 30 min; total running time 66 h.
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Fig. 2. Southern blots of genomic catfish DNA from different individual fish restricted with SfiI and separated by PFGE. The blot was sequentially hybridized with probes specific for the functional C located within the first H chain gene cluster (panel A) or with probes specific for the pseudo-C located within the second H chain gene cluster (panel B). The lane numbers 1–5 designate genomic DNA samples from fishes 9–12, and 7, respectively. The relative sizes of the fragments are shown in kb units on the left of the panels. The pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 25 s; final pulse time 70 s; total running time 26 h.
range in these fish from 370 to 530 kb. This result required additional mapping studies to clarify this difference and closely resolve the physical map of the catfish IgH locus. 3.3. Physical map of the IgH locus of the channel catfish In earlier sequencing studies, BssHII sites were identified within the C1 of both gene clusters. These recognition sites proved important in subsequent analyses. The cluster-specific C probes were derived from regions of
the clusters that are downstream from the BssHII site (see Section 2). Two additional probes were derived that were required for these mapping studies. The first, designated JH, hybridizes with JH segments located upstream of the C BssHII site in both gene clusters. The second probe, designated 5 -JH, is a cluster-specific probe corresponding to a region located immediately upstream of the JH1 segment within the first H chain gene cluster. Genomic DNA was partially restricted with SfiI, BssHII, or with both SfiI and BssHII, and Southern blots were sequentially hybridized
Fig. 3. Partial restriction map of the channel catfish IgH locus showing the relative locations of the duplicated C regions within the first and second H chain gene clusters of four individual channel catfish. The numbers designating the individual fish are shown on the left. The abbreviations for the restriction enzymes used in these analyses are: E: EagI, S: SfiI. B: BssHII; N: NruI. The functional C within the first H chain cluster is indicated as a solid box, the pseudogene C within the second H chain cluster is indicated as a stippled box. The exact location of the C regions within the indicated SfiI restriction fragments in fishes 10 and 12 was not determined, but is depicted in positions consistent with that experimentally determined in fishes 8 and 7.
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with the cluster-specific C probes, J probes (JH and 5 -JH), and VH family-specific probes. Separate analyses with genomic DNA from two fish (fishes 7 and 8) were used in these studies and representative results from fish 7 are shown in Fig. 4. As seen above, the functional C in fish 7 is contained on a 290 kb SfiI fragment (or a 300 kb SfiI fragment in fish 8) that is flanked downstream by a 240 and a 170 kb SfiI fragments
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(Fig. 4, panel A). On the BssHII map the functional C is contained on a 145 kb BssHII fragment and flanked downstream by a 75 kb BssHII fragment. Neither of these BssHII fragments hybridized with any of the VH family-specific probes; a result which indicates that VH fragments are not located downstream of the functional C. This important point was confirmed using double digests of SfiI with NruI or EagI (Fig. 5). EagI and NruI sites mapped within 30 kb of each other and flanked the distal SfiI site that contains the functional C (Fig. 3). Thus, these results, coupled with the above data that showed the IgH locus is contained within a 1.45 Mb EagI or a 1.55 Mb NruI fragment, indicate that catfish VH segments are only located upstream of the 145 kb BssHII fragment that contains the functional C. The analysis of the overlapping BssHII and SfiI restriction maps of the IgH locus in fishes 7 and 8 proved revealing. Comparison of the restriction maps, shows that only five of the seven (fish 7) or eight (fish 8) SfiI sites identified in the IgH locus were conserved in both fish. Similarly, eight of the 10 (fish 8) or 12 (fish 7) BssHII sites appeared conserved in both fish. However, even with these variations, which likely represent restriction site polymorphisms (see Section 4), the total distance between the BssHII site in the C1 exon of the first cluster and the BssHII site in the C1 pseudogene of the second gene cluster was similar. In fish 7, this distance was 715 kb, and in fish 8, this distance was 735 kb, for an average distance of 725 kb. These results also showed that the gene clusters are in the same relative transcription orientation. 3.4. Location of VH genes within the catfish IgH locus
Fig. 4. Southern blots of genomic catfish DNA (fish 7) restricted with SfiI and/or BssHII and separated by PFGE. Lane 1, SfiI restricted DNA; lane 2, BssHII restricted DNA; and lane 3, DNA restricted with both SfiI and BssHII. The Southern blot was sequentially hybridized with probes specific for defined regions within the first and second H chain gene clusters. The various panels (A–F) represent the blot hybridized with different probes: panel A, C probe specific for the functional gene in the first gene cluster; panel B, C probe specific for the pseudogene in the second gene cluster; panel C, probe specific for JH segments found in both the first and second H chain gene clusters. The blots were also sequentially hybridized with probes specific for each of the catfish VH families and representative results from three of these blots are shown: panel D, VH2; panel E, VH3; and panel F, VH5. The relative sizes of the fragments are shown in kb units on the left of the panels. The pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 4 s; final pulse time 40 s; total running time 18 h.
As determined above, VH segments do not appear to be present downstream of the functional C. Thus, the last question addressed in this study was to determine the relative location of VH gene segments within the IgH locus. The favorable distribution of different sized BssHII and SfiI restriction fragments in fish 7 allowed several conclusions to be made. Hybridization studies with the family-specific probes showed that VH segments are located on the 290 kb SfiI fragment that contains the functional C as well on the large 530 kb SfiI fragment that contains the pseudo-C. VH segments are also located on each of the SfiI fragments between these fragments. The large 530 kb SfiI fragment which contains the pseudo-C as well as the adjacent downstream 120 kb SfiI fragment were positive for each of the VH families. The 75 and 290 kb SfiI fragments were positive for each of the VH families with the following exceptions: the 75 kb fragment did not appear to hybridize with probes for VH3, VH4, and VH7, and the 290 kb fragment did not appear to hybridize with probes for VH1 and VH6. It was not feasible to further extend exposure times and it is left unresolved whether these specific fragments may contain a limited number of members representing these VH families. Hybridization of the BssHII fragments with the VH family-specific probes also showed that VH segments appear to be present on each of the BssHII fragments located
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Fig. 5. Southern blots of genomic catfish DNA (fish 8) restricted with SfiI alone or in combination with EagI, or NruI and separated by PFGE. The probes used for hybridization were specific for the functional C in the first H chain gene cluster (panel A) and for the C pseudogene in the second gene cluster (panel B). Lane 1: DNA restricted with SfiI. Lane 2: DNA restricted with SfiI and EagI. Lane 3: DNA restricted with SfiI and NruI. Fragment sizes are indicated in kb units on the left of the panels. Pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 4 s; final pulse time 70 s; total running time 24 h.
between the pseudo-C and the functional C. VH segments are also located upstream of the C pseudogene. The 150 kb BssHII fragment located immediately upstream of the pseudo C was positive for each of the VH families except for VH4 and VH7. It could not be determined if VH segments resided on the adjacent upstream 120 kb BssHII fragment because this fragment was defined from a 270 kb BssHII partial restriction product. Thus, it can be concluded that VH segments representing multiple gene families are widely dispersed within the 725 kb region that extends between the C regions of the first and second H chain gene clusters. VH segments are located upstream of the C pseudogene, but appear to extend no further than 270 kb upstream.
4. Discussion The results of this study have shown that the channel catfish IgH locus represents a very different organizational pattern of V, D, J, and C region segments from that previously known in other vertebrates. The catfish IgH locus is a composite represented by two different clusters of Ig genes referred to as the first and second gene clusters. The first cluster contains multiple VH segments, a DH locus represented by at least three functional DH segments, a JH locus containing nine functional JH segments, and a single functional copy of C. The second gene cluster is represented by multiple VH segments, a germline-joined VDJ, a single JH segment and a pseudogene C. When the second gene cluster was initially characterized, sequence determination showed that there was extensive homology between the two gene clusters. The homology initiated within the J region of the germline-joined VDJ and extended down-
stream to included the pseudogene C exons (Ghaffari and Lobb, 1999). These earlier studies also identified a transposon of the Tc1/mariner class of transposable elements that had integrated downstream of the pseudo C2 exon. This result indicated that transposition likely contributed to the disruption of the C in the second gene cluster. Transposition events may have also led to the initial duplication event. Although alternate hypotheses must be considered, such as unequal homologous recombination, it is nonetheless reasonable to postulate that the presence of the germline-joined VDJ indicates that germline recombination event(s) likely occurred after the duplication. Germline recombination would lead to deletion of germline segments between the recombined V, D, and J germline segments based upon earlier studies which have shown that the characterized D, J, and C segments within the first gene cluster are all in the same transcriptional orientation. As such, the second gene cluster may represent only a portion of the gene segments that arose from the initial duplication event. Duplication of extended regions of Ig genes has been identified in other vertebrates. For example, in man large duplications have occurred within the kappa locus (Huber et al., 1993; Schäble and Zachau, 1993). These studies indicate that duplication of extensive genomic regions of Ig genes is a repeated pattern during vertebrate phylogeny. The results from this study indicate that the first and second gene clusters are closely linked. The initial data showed that both clusters are located on a single, large AscI (4.0 Mb) or NotI (5.7 Mb) fragment, and by empirically using a series of other restriction enzymes, the IgH locus was identified on a 1.45 Mb EagI fragment. This identical-sized fragment hybridized with probes specific for the C regions within both clusters as well as with JH, and VH specific probes representing seven different catfish VH families. The
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organization of the gene clusters within this fragment was characterized using SfiI, and BssHII, and several general features of the catfish IgH locus were identified. Parallel mapping studies using genomic DNA obtained from different individual outbred animals indicated that the size of the IgH locus was similar between individual fish. The cumulative mapped distance between the fragments that contain the C in the first and second gene clusters in these fish varied by about 3–5%. Differences, however, were identified in the locations of some of the SfiI as well as BssHII sites in the IgH maps of these different fish. Although these differences most likely reflect restriction fragment length polymorphisms, the restriction conditions employed produced partial rather than complete digestion, and as such, the accessibility of these restriction sites may vary. This variation may also reflect differences in methylation of the CpG sequences within the restriction recognition sites (Smith and Cantor, 1989). Polymorphisms, as well as insertions and deletions, are widely known to occur within the IgH locus of other vertebrates. For example, in man duplicated and deleted haplotypes within just the IgH constant region account for up to 6% variation in the size of this region (Brusco et al., 1997). Polymorphisms are also often associated with the cleavage sites of most enzymes that yield large DNA fragments (Smith and Cantor, 1989). These studies also found that VH segments could not be detected downstream of the functional C; a result consistent with known organizational patterns of VH segments in the IgH locus of higher vertebrates (Matsuda et al., 1998). In addition, there was no evidence obtained in this study that VH segments were located in the genome outside of the mapped locus. These latter results were somewhat unexpected. In the shark Heterodontus, it is estimated that there are 200 or more gene clusters with V, D, J, and C region segments in each cluster (Kokubu et al., 1988). In addition, in situ hybridization approaches with the VH clusters in rays have shown that these clusters are widely dispersed and located on multiple chromosomes (Anderson et al., 1994). VH pseudogenes often retain major characteristic structural features that would, for example allow them to be detected by hybridization approaches. This structural conservation has led to hypotheses that VH pseudogenes serve an important role in the development of the immune repertoire by serving as a reservoir of sequences that are more freely able to diverge (reviewed in Vargas-Madrazo et al., 1995; Kawasaki et al., 2000). Yet at later points in time, through gene conversion (McCormack et al., 1991; Ota and Nei, 1994; Knight and Barrington, 1998) and other events such as molecular drive (Dover and Strachan, 1987), pseudogenes become reintegrated into the functional VH gene pool. The catfish VH pseudogenes that have been sequenced were initially detected by hybridization with family-specific probes and these segments retained readily recognizable structural features (Ventura-Holman et al., 1994). Hence, these results suggest that if the genomic organization of H chain genes in early vertebrate phylogeny was in multiple,
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chromosomally-dispersed clusters of gene segments, then VH gene segments outside of the mapped IgH region should have been detected in this bony fish. This was not the case, and hence these results suggest that the gene arrangement of H chain gene segments in primitive vertebrates was not in clusters, but that multiple clusters such as those identified in sharks and rays arose by subsequent duplication and translocation events. The mapping studies showed that the distance between the duplicated C1 regions was ∼725 kb. VH gene segments representing multiple gene families are located within the SfiI as well as the BssHII restriction fragments that separate the duplicated C regions. VH segments are present in the ∼110 kb BssHII fragment which contains the DH and JH segments associated with the first gene cluster. Additional studies were conducted to determine the distance from the DH segments to the upstream proximal VH segment. PFGE studies with PacI, NheI, and NruI genomic digests, however, were unable to adequately resolve this question due to the number of VH positive fragments within the 9–100 kb range (data not shown). Thus, the distance between the DH segments and the upstream proximal VH segments within the first cluster could not be further resolved. Multiple VH segments representing different families are, however, located immediately upstream of the germline-joined VDJ in the second gene cluster. Their presence offers interesting hypotheses of the potential impact this organizational pattern may have on the repertoire of expressed VH segments. Based upon the genomic restriction maps and the hybridization data obtained, the catfish IgH locus is conservatively estimated to be no greater than 1 Mb in length. The locus may be as small as 750–800 kb depending upon the number of VH segments that extend upstream of the pseudogene C in the second gene cluster and whether there are additional uncharacterized elements downstream of the functional C, and the C region exons that appear related to ␦ (Wilson et al., 1997). This estimate indicates that even with the internal duplication, the catfish IgH locus is smaller than that known in mammals. For example, in man just the VH locus, which contains 123 functional and pseudogene VH segments, spans about a 950 kb region located upstream of JH1 (Matsuda et al., 1998). In lower vertebrates, the size of the IgH locus is generally not known except in the chicken where a cluster of 80–100 VH pseudogene segments spans a region of 60–80 kb upstream of the single functional VH gene (Reynaud et al., 1987). Earlier studies had suggested that the IgH locus of bony fish appears relatively compact (Ghaffari and Lobb, 1992; Ventura-Holman et al., 1994), and this study supports this conclusion. In conclusion, the catfish IgH locus represents the product of a major duplication event resulting in two closely linked clusters of H chain gene segments. The clusters are in the same relative transcriptional orientation and the second, duplicated gene cluster appears to have undergone secondary gene rearrangement to form a germline rearranged VDJ. VH
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gene segments representing multiple gene families are located upstream of the VDJ as well as in each of the major restriction fragments that separate the pseudogene C exons in the second cluster from the functional C in the first gene cluster. VH gene segments were not detected downstream of the functional C within the first gene cluster nor were VH segments detected outside of the characterized locus. The continuing analysis of the divergence patterns of the gene segments associated with these clusters should lead to new insights as the relationship of gene organization, structure and repertoire function is examined. Acknowledgements This work was supported by Grant AI23052 from the National Institutes of Health. References Anderson, M., Amemiya, C., Luer, C., Litman, R., Rast, J., Niimura, Y., Litman, G., 1994. Complete genomic sequence and patterns of transcription of a member of an unusual family of closely related, chromosomally-dispersed Ig gene clusters in Raja. Int. Immunol. 6, 1661–1670. Brodeur, P.H., Osman, G.E., Mackle, J.J., Lalor, T.M., 1988. The organization of the mouse Igh-V locus: dispersion, interspersion, and the evolution of VH gene family clusters. J. Exp. Med. 168, 2261– 2278. Brusco, A., Cinque, F., Saviozzi, S., Boccazzi, C., DeMarchi, M., Carbonara, A.O., 1997. The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes. Hum. Genet. 100, 84–89. Dover, G.A., Strachan, T., 1987. Molecular drive in the evolution of the immune superfamily of genes: the initiation and spread of novelty. In: Kelsoe, G., Schulze, D.H. (Eds.), Evolution and Vertebrate Immunity: The Antigen Receptor and MHC Gene Families. University of Texas Press, Austin, TX, p. 15. Ghaffari, S.H., Lobb, C.J., 1989a. Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family. J. Immunol. 142, 1356–1365. Ghaffari, S.H., Lobb, C.J., 1989b. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains. J. Immunol. 143, 2730–2739. Ghaffari, S.H., Lobb, C.J., 1991. Heavy chain variable region gene families evolved early in phylogeny Ig complexity in fish. J. Immunol. 146, 1037–1046. Ghaffari, S.H., Lobb, C.J., 1992. Organization of immunoglobulin heavy chain constant and joining region genes in the channel catfish. Mol. Immunol. 29, 151–159. Ghaffari, S.H., Lobb, C.J., 1993. Structure and genomic organization of immunoglobulin light chain in the channel catfish. An unusual genomic organizational pattern of segmental genes. J. Immunol. 151, 6900– 6912. Ghaffari, S.H., Lobb, C.J., 1997. Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish. J. Immunol. 159, 250–258. Ghaffari, S.H., Lobb, C.J., 1999. Structure and genomic organization of a second cluster of immunoglobulin heavy chain gene segments in the channel catfish. J. Immunol. 162, 1519–1529.
Hayman, J.R., Ghaffari, S.H., Lobb, C.J., 1993. Heavy chain joining region segments of the channel catfish. Genomic organization and phylogenetic implications. J. Immunol. 151, 3587–3596. Hayman, J.R., Lobb, C.J., 2000. Heavy chain diversity region segments of the channel catfish: structure, organization, expression, and phylogenetic implications. J. Immunol. 164, 1916–1924. Huber, C., Huber, E., Lautner-Riesks, A., Schäble, K.F., Zachau, H.G., 1993. The human immunoglobulin locus. Characterization of the partially duplicated L regions. Eur. J. Immunol. 23, 2860–2867. Kawasaki, K., Minoshima, S., Shimizu, N., 2000. Propagation and maintenance of the 119 human immunoglobulin V genes and pseudogenes during evolution. Mol. Dev. Evol. 288, 120–134. Knight, K.L., Barrington, R.A., 1998. Somatic diversification of IgH genes in rabbit. Immunol. Rev. 162, 37–47. Kokubu, F., Litman, R., Shamblott, M.J., Hinds, R., Litman, G.W., 1988. Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate. EMBO J. 7, 3413–3422. Lobb, C.J., 1985. Covalent structure and affinity of channel catfish anti-dinitrophenyl antibodies. Mol. Immunol. 22, 993–999. Matsuda, F., Bourvagnet, P., Miyata, T., Honjo, T., 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J. Exp. Med. 188, 2151–2162. McCormack, W.T., Tjoelker, L.W., Thompson, C.B., 1991. Avian B-cell development: generation of an immunoglobulin repertoire by gene conversion. Ann. Rev. Immunol. 9, 219–241. Ota, T., Nei, M., 1994. Evolution of immunoglobulin VH pseudogenes in chickens. Mol. Biol. Evol. 12, 94–102. Reynaud, C.-A., Anquez, V., Grimal, H., Weill, J.C., 1987. A hyperconversion mechanism generates the chicken light chain pre-immune repertoire. Cell 48, 379–388. Schäble, K.F., Zachau, H.G., 1993. The variable genes of the human immunoglobulin locus. Biol. Chem. Hoppe. Seyler. 374, 1001 1022. Schluter, S.F., Bernstein, R.M., Marchalonis, J.J., 1997. Molecular origins and evolution of immunoglobulin heavy-chain genes of jawed vertebrates. Immunol. Today 18, 543–549. Smith, C.L., Cantor, C.R., 1989. Evolving strategies for making physical maps of mammalian chromosomes. Genome 31, 1055–1058. Smith, C.L., Klco, S, Zhang, T.Y., Fang H., Oliva, R., Wang, D., Bremer, M., Lawrance, S., 1993. Analysis of megabase DNA using pulsed-field electrophoresis techniques. In: Adolph, K.W. (Ed.), Methods in Molecular Genetics, Vol. 2. Academic Press, San Diego, pp. 129–154. Tonegawa, S., 1983. Somatic diversification of antibody diversity. Nature 302, 575–581. Vargas-Madrazo, E., Almagro, J.C., Lara-Ochoa, F., 1995. Structural repertoire in VH pseudogenes of immunoglobulins: comparison with human germline genes and human amino acid sequences. J. Mol. Biol. 246, 74–81. Ventura-Holman, T., Jones, J.C., Ghaffari, S.H., Lobb, C.J., 1994. Structure and genomic organization of VH gene segments in the channel catfish: members of different VH gene families are interspersed and closely linked. Mol. Immunol. 31, 823–832. Ventura-Holman, T., Ghaffari, S.H., Lobb, C.J., 1996. Characterization of a seventh family of immunoglobulin heavy chain VH gene segments in the channel catfish, Ictalurus punctatus. Eur. J. Immunogenet. 23, 7–14. Ventura-Holman, T., Lobb, C.J., 1995. High molecular weight DNA from nucleated erythrocytes for use in pulsed-field gel electrophoresis (PFGE). Genet. Anal. 12, 101–103. Wilson, M., Bengtén, E., Miller, N.W., Clem, L., DuPasquier, W.L., Warr, G.W., 1997. A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc. Natl. Acad. Sci. U.S.A. 94, 4593–4597. Yancopoulos, G., Alt, F., 1986. Regulation of the assembly and expression of variable region genes. Ann. Rev. Immunol. 4, 339–368.
Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters Tereza Ventura-Holman, Craig J. Lobb∗ Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA Received 1 July 2001; accepted 10 August 2001
Abstract Two structurally-related genomic clusters of catfish immunoglobulin heavy chain gene segments are known. The first gene cluster contains DH and JH segments, as well as the C region exons encoding the functional C. The second gene cluster contains multiple VH gene segments representing different VH families, a germline-joined VDJ, a single JH segment, and at least two pseudogene C exons. It was not known whether these gene clusters were linked, nor was the organization or the location of VH segments associated within the first gene cluster known. Pulsed-field gel electrophoresis studies have been used to determine the structural organization of these gene clusters. Restriction mapping studies show that the two gene clusters are closely linked; the second gene cluster is located upstream from the first with the C regions within the clusters separated by about 725 kb. The clusters are in the same relative transcriptional orientation, and the results indicate that the complete IgH locus spans no more than 1000 kb and may be as small as 750–800 kb. VH gene segments are located both upstream and downstream of the pseudo-C exons; however, no VH gene segments that hybridized with the VH specific probes were detected downstream of the functional C. These studies coupled with earlier sequence analyses indicate that the catfish IgH locus arose from a massive internal duplication event. Subsequent gene rearrangement within the duplicated cluster likely resulted in the presence of the germline VDJ and the deletion of intervening V, D and J segments. Transposition by a member of the Tc1/mariner family of transposable elements appears to have led to the disruption of the duplicated C. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Antibodies; Molecular biology; Comparative immunology; Gene evolution
1. Introduction Although the general structure of immunoglobulin V, D, J and C region segments has been conserved during vertebrate phylogeny, the evolution of the genomic organization and utilization patterns of these gene segments has been a dynamic process. The most familiar form of genomic organization is that represented in humans and mice, wherein various functional VH segments representing different VH families are located upstream from DH segments that are, in turn, located upstream of JH and C region segments (Tonegawa, 1983; Brodeur et al., 1988; Yancopoulos and Alt, 1986; Matsuda et al., 1998). A second organizational pattern is represented in chickens. The locus resembles that of humans and mice in that there are multiple VH segments upstream of DH, JH, and CH segments; however, in this case, only a single VH segment is functional. The other VH segments are pseudogenes, and their contribution to the diversity of the Ig Abbreviations: H, heavy chain of immunoglobulin; L, light chain of immunoglobulin; Mb, megabase; PFGE, pulsed-field gel electrophoresis ∗ Tel.: +1-601-984-1700; fax: +1-601-984-1708. E-mail address: [email protected] (C.J. Lobb).
repertoire is manifested only through gene conversion mechanisms (Reynaud et al., 1987; McCormack et al., 1991). A third organizational pattern is that represented in sharks, where there are multiple clusters of Ig gene segments. Within each cluster there are V, D, J, and C segments, and gene rearrangement appears restricted to the segments within individual clusters (Kokubu et al., 1988; Schluter et al., 1997). Recent studies have shown that the organization of Ig genes in channel catfish reflects a phylogenetic “anthology” of different genomic organizational patterns of segmental Ig genes. The L chains, represented by two different classes, are both organized in multiple gene clusters wherein V, J and C gene segments reside within each cluster. The VL segments are in inverted transcriptional orientation relative to the J and C segments, which indicates that VL segments likely undergo rearrangement to JL–CL segments by inversion (Ghaffari and Lobb, 1993; Ghaffari and Lobb, 1997). In contrast, the H chain locus contains only a single functional C gene that encodes the four domain C region of the predominant serum antibody and Ig (Ghaffari and Lobb, 1989a,b; Lobb, 1985). The JH locus contains nine functional JH segments that are tightly clustered within a 2.2 kb region located immediately upstream from C (Hayman
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et al., 1993). The DH locus was recently identified through the examination of circular recombination excision products which proved that germline D–J recombination can result in deletion events. This locus contains at least three different DH segments that represent single member gene families, and is located about 8.6 kb upstream of the JH locus (Hayman and Lobb, 2000). In addition, it has been shown that VH gene families extensively diverged at the level of the bony fish. Southern blot studies indicate an extensive genomic VH repertoire consisting of at least seven VH gene families which represent >100 different gene segments (reviewed in Ventura-Holman et al., 1996). Examination of lambda genomic libraries indicate that members of the different gene families are interspersed with one another and that VH segments are closely linked; the average distance between segments is about 3.5 kb (Ventura-Holman et al., 1994). These results indicate that the structure, genomic organization, and recombination patterns of H chain genes that are typically associated with higher vertebrates evolved early in phylogeny at the level of the bony fish. Recently, a second cluster of channel catfish H chain gene segments was defined (Ghaffari and Lobb, 1999). The characterized 25 kb region of this second H chain cluster consists of multiple VH segments, representing different VH families located upstream of a germline-joined VDJ segment. The VDJ has a split leader sequence and a single open reading frame consistent with that known in expressed members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene C exons, C1 and C2. These exons are multiply crippled with RNA splice sites destroyed, and open reading frames interrupted by termination codons, insertions, and/or deletions. A 10.8 kb sequenced region was readily aligned with homologous JH and C exons in the first gene cluster, which indicated that this second cluster of H chain gene segments shared a close structural relationship with gene segments in the first cluster. With these results it was essential to characterize the genomic organization of these two H chain gene clusters. This report characterizes this relationship and provides new insights into the mechanisms underlying early evolutionary patterns of the IgH locus.
2. Materials and methods 2.1. Preparation and restriction of genomic DNA in agarose blocks for analysis by pulsed-field gel electrophoresis Erythrocytes from individual adult channel catfish (Ictalurus punctatus) were used as the source of genomic DNA for PFGE analyses. The genomic DNA agarose embedding procedure used in these studies is based on the procedure described by Smith et al. (1993), and modified as described in detail (Ventura-Holman and Lobb, 1995). Prior to restriction, agarose blocks containing 2–3 g of DNA were equili-
brated in three 15 min washes of the appropriate restriction buffer, and then incubated overnight with a maximum of 10 units of restriction enzyme/g DNA in 150 l of buffer at the recommended temperature (New England Biolabs, Beverly, MA). 2.2. PFGE running conditions: Southern blots, and hybridization conditions Molecular weight standards, prepared in agarose blocks by the manufacturer (Bio-Rad, Hercules, CA), were run in lanes adjacent to the restricted genomic DNA samples. The standards used in these experiments included: a lambda ladder for DNA fragment sizes ranging from 50 kb to 1 Mb; Saccharomyces cerevisiae chromosomes for sizes ranging from 200 kb to 2.2 Mb; and Hansenula wingei chromosomes for sizes ranging from 1 to 3.1 Mb. The molecular weight standards or restricted DNA was separated by agarose gel electrophoresis using a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad) using running conditions as described in the figure legends. For DNA fragments greater than 1 Mb in size, 0.8% agarose gels were prepared using 1X TAE in chromosomal grade agarose (Bio-Rad). For fragments less than 1 Mb, 1% gels in 0.5X TBE were prepared using pulsed-field certified agarose (Bio-Rad). The running buffer was the same as used to prepare the gel. The temperature of the running buffer was maintained at 14◦ C during electrophoresis. Following electrophoresis, the DNA was transferred to Zeta-Probe GT membranes (Bio-Rad) by capillary transfer, and cross-linked by UV irradiation as recommended by the manufacturer. The membranes were pre-washed in 3X SSC and 0.1% SDS at 65 ◦ C for 1 h and pre-hybridized for 6 h to overnight in 3X SSC, 5X Denhardt’s solution, 50% formamide and 0.1% SDS at 42 ◦ C. The blots were hybridized with radiolabeled probes in 3X SSC, 1X Denhardt’s solution, 50% formamide, and 0.2% SDS at 42 ◦ C overnight. The membranes were washed twice in 2X SSC, 0.1% SDS and four times in 0.2X SSC, 0.1% SDS at 65 ◦ C before autoradiography at −70 ◦ C (typically a 3-day exposure). For sequential hybridizations, the membranes were stripped of the radiolabeled probe by two 15 min washes in 0.5% SDS, 0.5X SSC. The boiling solution was poured over the blots and incubated at 85 ◦ C with shaking. Probes used in these studies included the following VH family-specific probes derived from cDNA clones previously described (Ghaffari and Lobb, 1991; Ventura-Holman et al., 1994; Ventura-Holman et al., 1996): a 300 bp PstI fragment from NG70 (VH1); a 378 bp PvuII fragment from NG41 (VH2); a 266 bp PstI fragment from NG54 (VH3); a 260 bp PvuII–BstEII fragment from NG10 (VH4); a 364 bp PvuII–BamHI fragment from NG66 (VH5); a 457 bp fragment from clone 10.4 (VH6); a 221 bp SstI–AvaI fragment from clone vh5e (VH7). The following probes were also used: a 778 bp EcoRI cDNA restriction fragment (containing the C3 and C4 domains of the functional C); a
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1.5 kb PstI–SstI restriction fragment of genomic clone C2, corresponding to a region (intron) located between the pseudogene C1 and C2 exons of the second H chain cluster (Ghaffari and Lobb, 1999); a 1.2 kb ClaI restriction fragment located within the cluster of JH segments found upstream of the functional C (designated as the JH probe, Hayman et al., 1993); and a 5.6 kb EcoRI restriction fragment located upstream of the JH locus in the first gene cluster (designated as the 5 -JH probe, Hayman and Lobb, 2000). Probes were radiolabeled with [32 P]dCTP to a specific activity of approximately 108 –109 cpm/g of DNA using either a multiprime or megaprime random priming kit (Amersham, Arlington Heights, IL).
3. Results 3.1. Linkage of the first and second H chain clusters Studies were initially undertaken to determine if the two catfish H chain gene clusters were linked on the same large genomic restriction fragments. The approach that was taken was to derive specific probes from C regions within each gene cluster and hybridize these probes to genomic restriction fragments that had been separated by PFGE. Agarose-embedded genomic DNA was restricted with either AscI or NotI, and following PFGE, the blot was hybridized with the cluster-specific C probes. These results showed that both probes hybridized with the same sized genomic fragment, an AscI fragment of 4.0 Mb or a NotI fragment of 5.7 Mb. The blots were sequentially hybridized with probes specific for each of the seven VH families, and in each case this identical-sized fragment was the only band that hybridized with these probes (data not shown). The linkage distance of the H chain gene clusters was narrowed with the use of additional 6 bp recognition site restriction enzymes. Genomic Southern blots of DNA restricted with NruI or EagI and hybridized with the cluster-specific C probes yielded single fragments of 1.55 or 1.45 Mb, respectively. When the blots were sequentially hybridized with specific probes for each of the seven VH families, these
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identical-sized fragments were the only fragments that were detected (Fig. 1). Double digests of genomic DNA restricted with both NruI and EagI revealed only the 1.45 Mb fragment when blots were hybridized with these probes. These combined analyses indicate that the two gene clusters are linked within 1.45 Mb and that all VH segments detected by these family-specific probes are contained on this size of fragment. 3.2. Linkage distance between the first and second H chain clusters The organization of the two H chain gene clusters within the 1.45 Mb region was further defined with the enzyme SfiI. The approach that was taken was to use restriction conditions that yielded partial digests of genomic DNA within the 100–1000 kb range. Southern blots of the PFGE-separated fragments were then sequentially hybridized with the cluster-specific C probes, and the restriction map of the overlapping fragments was determined. Fig. 2 shows the hybridization patterns of SfiI restricted DNA obtained from five individual outbred channel catfish. Three of these fish (fishes 9, 11 and 7, lanes 1, 3, and 5, respectively) yielded essentially the same hybridization patterns with the cluster-specific probes. Genomic DNA from the other two fish (fishes 10 and 12, lanes 2 and 4, respectively) yielded restriction patterns that were distinct from the other fish as well as somewhat different from each other. Representative restriction maps of these overlapping fragments obtained from these SfiI digests from these fish are shown in Fig. 3. These analyses showed that the SfiI fragment that contained the pseudo-C was closely linked to the fragment that contained that functional C. Two internal SfiI fragments separated the SfiI fragments defined by these cluster-specific C probes. The combined size of these two fragments ranged in size from 150–195 kb. The SfiI fragment that contained the functional C in fishes 9, 10, and 12 was about 310 or about 290 kb in fishes 11 and 7, respectively. In each case, this fragment was flanked downstream by a 240 kb SfiI fragment. However, the size of the SfiI fragments that hybridized with the specific probes for the pseudo C exhibited a size
Fig. 1. Southern blots of genomic catfish DNA (fish 4) restricted with EagI (lane 1) or NruI (lane 2) and separated by PFGE. The probes used for hybridization were specific for the C located within the first H chain gene cluster (panel A) or probes specific for the pseudo-C located within the second H chain gene cluster (panel B). Probes specific for each of the VH families 1–7 were sequentially hybridized with the blot and the results were identical. Representative results using the probe for the VH3 family are shown (panel C). The relative sizes of the fragments are shown in Mb units on the left of the panels. The pulsed-field electrophoresis parameters were: 0.8% agarose gel; 1X TAE; 2 V/cm; 106◦ included angle; initial pulse time 2 min; final pulse time 30 min; total running time 66 h.
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Fig. 2. Southern blots of genomic catfish DNA from different individual fish restricted with SfiI and separated by PFGE. The blot was sequentially hybridized with probes specific for the functional C located within the first H chain gene cluster (panel A) or with probes specific for the pseudo-C located within the second H chain gene cluster (panel B). The lane numbers 1–5 designate genomic DNA samples from fishes 9–12, and 7, respectively. The relative sizes of the fragments are shown in kb units on the left of the panels. The pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 25 s; final pulse time 70 s; total running time 26 h.
range in these fish from 370 to 530 kb. This result required additional mapping studies to clarify this difference and closely resolve the physical map of the catfish IgH locus. 3.3. Physical map of the IgH locus of the channel catfish In earlier sequencing studies, BssHII sites were identified within the C1 of both gene clusters. These recognition sites proved important in subsequent analyses. The cluster-specific C probes were derived from regions of
the clusters that are downstream from the BssHII site (see Section 2). Two additional probes were derived that were required for these mapping studies. The first, designated JH, hybridizes with JH segments located upstream of the C BssHII site in both gene clusters. The second probe, designated 5 -JH, is a cluster-specific probe corresponding to a region located immediately upstream of the JH1 segment within the first H chain gene cluster. Genomic DNA was partially restricted with SfiI, BssHII, or with both SfiI and BssHII, and Southern blots were sequentially hybridized
Fig. 3. Partial restriction map of the channel catfish IgH locus showing the relative locations of the duplicated C regions within the first and second H chain gene clusters of four individual channel catfish. The numbers designating the individual fish are shown on the left. The abbreviations for the restriction enzymes used in these analyses are: E: EagI, S: SfiI. B: BssHII; N: NruI. The functional C within the first H chain cluster is indicated as a solid box, the pseudogene C within the second H chain cluster is indicated as a stippled box. The exact location of the C regions within the indicated SfiI restriction fragments in fishes 10 and 12 was not determined, but is depicted in positions consistent with that experimentally determined in fishes 8 and 7.
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with the cluster-specific C probes, J probes (JH and 5 -JH), and VH family-specific probes. Separate analyses with genomic DNA from two fish (fishes 7 and 8) were used in these studies and representative results from fish 7 are shown in Fig. 4. As seen above, the functional C in fish 7 is contained on a 290 kb SfiI fragment (or a 300 kb SfiI fragment in fish 8) that is flanked downstream by a 240 and a 170 kb SfiI fragments
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(Fig. 4, panel A). On the BssHII map the functional C is contained on a 145 kb BssHII fragment and flanked downstream by a 75 kb BssHII fragment. Neither of these BssHII fragments hybridized with any of the VH family-specific probes; a result which indicates that VH fragments are not located downstream of the functional C. This important point was confirmed using double digests of SfiI with NruI or EagI (Fig. 5). EagI and NruI sites mapped within 30 kb of each other and flanked the distal SfiI site that contains the functional C (Fig. 3). Thus, these results, coupled with the above data that showed the IgH locus is contained within a 1.45 Mb EagI or a 1.55 Mb NruI fragment, indicate that catfish VH segments are only located upstream of the 145 kb BssHII fragment that contains the functional C. The analysis of the overlapping BssHII and SfiI restriction maps of the IgH locus in fishes 7 and 8 proved revealing. Comparison of the restriction maps, shows that only five of the seven (fish 7) or eight (fish 8) SfiI sites identified in the IgH locus were conserved in both fish. Similarly, eight of the 10 (fish 8) or 12 (fish 7) BssHII sites appeared conserved in both fish. However, even with these variations, which likely represent restriction site polymorphisms (see Section 4), the total distance between the BssHII site in the C1 exon of the first cluster and the BssHII site in the C1 pseudogene of the second gene cluster was similar. In fish 7, this distance was 715 kb, and in fish 8, this distance was 735 kb, for an average distance of 725 kb. These results also showed that the gene clusters are in the same relative transcription orientation. 3.4. Location of VH genes within the catfish IgH locus
Fig. 4. Southern blots of genomic catfish DNA (fish 7) restricted with SfiI and/or BssHII and separated by PFGE. Lane 1, SfiI restricted DNA; lane 2, BssHII restricted DNA; and lane 3, DNA restricted with both SfiI and BssHII. The Southern blot was sequentially hybridized with probes specific for defined regions within the first and second H chain gene clusters. The various panels (A–F) represent the blot hybridized with different probes: panel A, C probe specific for the functional gene in the first gene cluster; panel B, C probe specific for the pseudogene in the second gene cluster; panel C, probe specific for JH segments found in both the first and second H chain gene clusters. The blots were also sequentially hybridized with probes specific for each of the catfish VH families and representative results from three of these blots are shown: panel D, VH2; panel E, VH3; and panel F, VH5. The relative sizes of the fragments are shown in kb units on the left of the panels. The pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 4 s; final pulse time 40 s; total running time 18 h.
As determined above, VH segments do not appear to be present downstream of the functional C. Thus, the last question addressed in this study was to determine the relative location of VH gene segments within the IgH locus. The favorable distribution of different sized BssHII and SfiI restriction fragments in fish 7 allowed several conclusions to be made. Hybridization studies with the family-specific probes showed that VH segments are located on the 290 kb SfiI fragment that contains the functional C as well on the large 530 kb SfiI fragment that contains the pseudo-C. VH segments are also located on each of the SfiI fragments between these fragments. The large 530 kb SfiI fragment which contains the pseudo-C as well as the adjacent downstream 120 kb SfiI fragment were positive for each of the VH families. The 75 and 290 kb SfiI fragments were positive for each of the VH families with the following exceptions: the 75 kb fragment did not appear to hybridize with probes for VH3, VH4, and VH7, and the 290 kb fragment did not appear to hybridize with probes for VH1 and VH6. It was not feasible to further extend exposure times and it is left unresolved whether these specific fragments may contain a limited number of members representing these VH families. Hybridization of the BssHII fragments with the VH family-specific probes also showed that VH segments appear to be present on each of the BssHII fragments located
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Fig. 5. Southern blots of genomic catfish DNA (fish 8) restricted with SfiI alone or in combination with EagI, or NruI and separated by PFGE. The probes used for hybridization were specific for the functional C in the first H chain gene cluster (panel A) and for the C pseudogene in the second gene cluster (panel B). Lane 1: DNA restricted with SfiI. Lane 2: DNA restricted with SfiI and EagI. Lane 3: DNA restricted with SfiI and NruI. Fragment sizes are indicated in kb units on the left of the panels. Pulsed-field electrophoresis parameters were: 1% agarose gel; 0.5X TBE; 6 V/cm; 120◦ included angle; initial pulse time 4 s; final pulse time 70 s; total running time 24 h.
between the pseudo-C and the functional C. VH segments are also located upstream of the C pseudogene. The 150 kb BssHII fragment located immediately upstream of the pseudo C was positive for each of the VH families except for VH4 and VH7. It could not be determined if VH segments resided on the adjacent upstream 120 kb BssHII fragment because this fragment was defined from a 270 kb BssHII partial restriction product. Thus, it can be concluded that VH segments representing multiple gene families are widely dispersed within the 725 kb region that extends between the C regions of the first and second H chain gene clusters. VH segments are located upstream of the C pseudogene, but appear to extend no further than 270 kb upstream.
4. Discussion The results of this study have shown that the channel catfish IgH locus represents a very different organizational pattern of V, D, J, and C region segments from that previously known in other vertebrates. The catfish IgH locus is a composite represented by two different clusters of Ig genes referred to as the first and second gene clusters. The first cluster contains multiple VH segments, a DH locus represented by at least three functional DH segments, a JH locus containing nine functional JH segments, and a single functional copy of C. The second gene cluster is represented by multiple VH segments, a germline-joined VDJ, a single JH segment and a pseudogene C. When the second gene cluster was initially characterized, sequence determination showed that there was extensive homology between the two gene clusters. The homology initiated within the J region of the germline-joined VDJ and extended down-
stream to included the pseudogene C exons (Ghaffari and Lobb, 1999). These earlier studies also identified a transposon of the Tc1/mariner class of transposable elements that had integrated downstream of the pseudo C2 exon. This result indicated that transposition likely contributed to the disruption of the C in the second gene cluster. Transposition events may have also led to the initial duplication event. Although alternate hypotheses must be considered, such as unequal homologous recombination, it is nonetheless reasonable to postulate that the presence of the germline-joined VDJ indicates that germline recombination event(s) likely occurred after the duplication. Germline recombination would lead to deletion of germline segments between the recombined V, D, and J germline segments based upon earlier studies which have shown that the characterized D, J, and C segments within the first gene cluster are all in the same transcriptional orientation. As such, the second gene cluster may represent only a portion of the gene segments that arose from the initial duplication event. Duplication of extended regions of Ig genes has been identified in other vertebrates. For example, in man large duplications have occurred within the kappa locus (Huber et al., 1993; Schäble and Zachau, 1993). These studies indicate that duplication of extensive genomic regions of Ig genes is a repeated pattern during vertebrate phylogeny. The results from this study indicate that the first and second gene clusters are closely linked. The initial data showed that both clusters are located on a single, large AscI (4.0 Mb) or NotI (5.7 Mb) fragment, and by empirically using a series of other restriction enzymes, the IgH locus was identified on a 1.45 Mb EagI fragment. This identical-sized fragment hybridized with probes specific for the C regions within both clusters as well as with JH, and VH specific probes representing seven different catfish VH families. The
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organization of the gene clusters within this fragment was characterized using SfiI, and BssHII, and several general features of the catfish IgH locus were identified. Parallel mapping studies using genomic DNA obtained from different individual outbred animals indicated that the size of the IgH locus was similar between individual fish. The cumulative mapped distance between the fragments that contain the C in the first and second gene clusters in these fish varied by about 3–5%. Differences, however, were identified in the locations of some of the SfiI as well as BssHII sites in the IgH maps of these different fish. Although these differences most likely reflect restriction fragment length polymorphisms, the restriction conditions employed produced partial rather than complete digestion, and as such, the accessibility of these restriction sites may vary. This variation may also reflect differences in methylation of the CpG sequences within the restriction recognition sites (Smith and Cantor, 1989). Polymorphisms, as well as insertions and deletions, are widely known to occur within the IgH locus of other vertebrates. For example, in man duplicated and deleted haplotypes within just the IgH constant region account for up to 6% variation in the size of this region (Brusco et al., 1997). Polymorphisms are also often associated with the cleavage sites of most enzymes that yield large DNA fragments (Smith and Cantor, 1989). These studies also found that VH segments could not be detected downstream of the functional C; a result consistent with known organizational patterns of VH segments in the IgH locus of higher vertebrates (Matsuda et al., 1998). In addition, there was no evidence obtained in this study that VH segments were located in the genome outside of the mapped locus. These latter results were somewhat unexpected. In the shark Heterodontus, it is estimated that there are 200 or more gene clusters with V, D, J, and C region segments in each cluster (Kokubu et al., 1988). In addition, in situ hybridization approaches with the VH clusters in rays have shown that these clusters are widely dispersed and located on multiple chromosomes (Anderson et al., 1994). VH pseudogenes often retain major characteristic structural features that would, for example allow them to be detected by hybridization approaches. This structural conservation has led to hypotheses that VH pseudogenes serve an important role in the development of the immune repertoire by serving as a reservoir of sequences that are more freely able to diverge (reviewed in Vargas-Madrazo et al., 1995; Kawasaki et al., 2000). Yet at later points in time, through gene conversion (McCormack et al., 1991; Ota and Nei, 1994; Knight and Barrington, 1998) and other events such as molecular drive (Dover and Strachan, 1987), pseudogenes become reintegrated into the functional VH gene pool. The catfish VH pseudogenes that have been sequenced were initially detected by hybridization with family-specific probes and these segments retained readily recognizable structural features (Ventura-Holman et al., 1994). Hence, these results suggest that if the genomic organization of H chain genes in early vertebrate phylogeny was in multiple,
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chromosomally-dispersed clusters of gene segments, then VH gene segments outside of the mapped IgH region should have been detected in this bony fish. This was not the case, and hence these results suggest that the gene arrangement of H chain gene segments in primitive vertebrates was not in clusters, but that multiple clusters such as those identified in sharks and rays arose by subsequent duplication and translocation events. The mapping studies showed that the distance between the duplicated C1 regions was ∼725 kb. VH gene segments representing multiple gene families are located within the SfiI as well as the BssHII restriction fragments that separate the duplicated C regions. VH segments are present in the ∼110 kb BssHII fragment which contains the DH and JH segments associated with the first gene cluster. Additional studies were conducted to determine the distance from the DH segments to the upstream proximal VH segment. PFGE studies with PacI, NheI, and NruI genomic digests, however, were unable to adequately resolve this question due to the number of VH positive fragments within the 9–100 kb range (data not shown). Thus, the distance between the DH segments and the upstream proximal VH segments within the first cluster could not be further resolved. Multiple VH segments representing different families are, however, located immediately upstream of the germline-joined VDJ in the second gene cluster. Their presence offers interesting hypotheses of the potential impact this organizational pattern may have on the repertoire of expressed VH segments. Based upon the genomic restriction maps and the hybridization data obtained, the catfish IgH locus is conservatively estimated to be no greater than 1 Mb in length. The locus may be as small as 750–800 kb depending upon the number of VH segments that extend upstream of the pseudogene C in the second gene cluster and whether there are additional uncharacterized elements downstream of the functional C, and the C region exons that appear related to ␦ (Wilson et al., 1997). This estimate indicates that even with the internal duplication, the catfish IgH locus is smaller than that known in mammals. For example, in man just the VH locus, which contains 123 functional and pseudogene VH segments, spans about a 950 kb region located upstream of JH1 (Matsuda et al., 1998). In lower vertebrates, the size of the IgH locus is generally not known except in the chicken where a cluster of 80–100 VH pseudogene segments spans a region of 60–80 kb upstream of the single functional VH gene (Reynaud et al., 1987). Earlier studies had suggested that the IgH locus of bony fish appears relatively compact (Ghaffari and Lobb, 1992; Ventura-Holman et al., 1994), and this study supports this conclusion. In conclusion, the catfish IgH locus represents the product of a major duplication event resulting in two closely linked clusters of H chain gene segments. The clusters are in the same relative transcriptional orientation and the second, duplicated gene cluster appears to have undergone secondary gene rearrangement to form a germline rearranged VDJ. VH
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gene segments representing multiple gene families are located upstream of the VDJ as well as in each of the major restriction fragments that separate the pseudogene C exons in the second cluster from the functional C in the first gene cluster. VH gene segments were not detected downstream of the functional C within the first gene cluster nor were VH segments detected outside of the characterized locus. The continuing analysis of the divergence patterns of the gene segments associated with these clusters should lead to new insights as the relationship of gene organization, structure and repertoire function is examined. Acknowledgements This work was supported by Grant AI23052 from the National Institutes of Health. References Anderson, M., Amemiya, C., Luer, C., Litman, R., Rast, J., Niimura, Y., Litman, G., 1994. Complete genomic sequence and patterns of transcription of a member of an unusual family of closely related, chromosomally-dispersed Ig gene clusters in Raja. Int. Immunol. 6, 1661–1670. Brodeur, P.H., Osman, G.E., Mackle, J.J., Lalor, T.M., 1988. The organization of the mouse Igh-V locus: dispersion, interspersion, and the evolution of VH gene family clusters. J. Exp. Med. 168, 2261– 2278. Brusco, A., Cinque, F., Saviozzi, S., Boccazzi, C., DeMarchi, M., Carbonara, A.O., 1997. The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes. Hum. Genet. 100, 84–89. Dover, G.A., Strachan, T., 1987. Molecular drive in the evolution of the immune superfamily of genes: the initiation and spread of novelty. In: Kelsoe, G., Schulze, D.H. (Eds.), Evolution and Vertebrate Immunity: The Antigen Receptor and MHC Gene Families. University of Texas Press, Austin, TX, p. 15. Ghaffari, S.H., Lobb, C.J., 1989a. Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family. J. Immunol. 142, 1356–1365. Ghaffari, S.H., Lobb, C.J., 1989b. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains. J. Immunol. 143, 2730–2739. Ghaffari, S.H., Lobb, C.J., 1991. Heavy chain variable region gene families evolved early in phylogeny Ig complexity in fish. J. Immunol. 146, 1037–1046. Ghaffari, S.H., Lobb, C.J., 1992. Organization of immunoglobulin heavy chain constant and joining region genes in the channel catfish. Mol. Immunol. 29, 151–159. Ghaffari, S.H., Lobb, C.J., 1993. Structure and genomic organization of immunoglobulin light chain in the channel catfish. An unusual genomic organizational pattern of segmental genes. J. Immunol. 151, 6900– 6912. Ghaffari, S.H., Lobb, C.J., 1997. Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish. J. Immunol. 159, 250–258. Ghaffari, S.H., Lobb, C.J., 1999. Structure and genomic organization of a second cluster of immunoglobulin heavy chain gene segments in the channel catfish. J. Immunol. 162, 1519–1529.
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