Heavy Chain Diversity Region Segments of the Channel Catfish: Structure, Organization, Expression and Phylogenetic Implications

This information is current as of December 1, 2011 J. Russell Hayman and Craig J. Lobb J Immunol 2000;164;1916-1924

References

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright 2000 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Heavy Chain Diversity Region Segments of the Channel Catsh: Structure, Organization, Expression and Phylogenetic Implications1,2
J. Russell Hayman and Craig J. Lobb3
Circular DNA, derived from lymphocytes of juvenile channel catsh, was used to construct libraries that were screened to identify the products of immunoglobulin DH-JH excision events. Clones were characterized that contained DH to JH recombination signal joints. The signal joints represented 23-bp recombination signal sequences (RSS) identical to germline JH segments that were adjacent to DH 12-bp RSS elements. DH anking regions within the clones were used to probe a genomic library. Three germline DH gene segments containing 1119 bp coding regions anked by 12-bp RSS elements with conserved heptamers and nonamers were identied. The DH locus is closely linked to the JH locus, and Southern blots indicate that the DH segments represent different single member gene families. Analysis of H chain cDNA shows that each germline DH segment was expressed in functional VDJ recombination events involving different JH segments and members of different VH families. Several aspects of CDR3 junctional diversity were evident, including deletion of coding region nucleotides, N- and P-region nucleotide additions, alternate DH reading frame utilization, and point mutations. Coding region motifs of catsh DH segments are phylogenetically conserved in some DH segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of DH segments typically associated with higher vertebrates evolved early in vertebrate phylogeny at the level of the bony sh. The Journal of Immunology, 2000, 164: 1916 1924. uring mammalian B cell development, H chain V region gene recombination occurs in two steps. Initial recombination between DH and JH gene segments forms the rearranged DJ, which subsequently recombines with a VH gene segment to form the rearranged VDJ. Recombination between gene segments is mediated by recombinase enzymes that recognized the recombination signal sequences (RSS)4 adjacent to the gene segment coding regions. Recombination may occur when one segment has a 23-bp spacer and the other has a 12-bp spacer (the 12/23 rule) (1 4). During recombination, sequence variation occurs as a result of junctional formation. Nucleotides at the ends of the coding regions may be deleted, and there may be insertion of nontemplated nucleotides (N-region additions). If nucleotides are not deleted from the coding region, nucleotides palindromic to the end of the coding region may also be added to the coding sequence (P-region additions). Through these mechanisms and in concert with somatic mutation, the inherent structural diversity of expressed H chain V regions arises (reviewed in Ref. 4). There are three apparent roles served by DH segments. Fundamentally DH segments serve to bridge VH and JH segments and are

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Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216 Received for publication June 29, 1999. Accepted for publication December 3, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 2

This work was supported by Grant AI23052 from the National Institutes of Health.

The sequences discussed in this paper have been entered into the GenBank database under the accession numbers AF161271AF161289.
3 Address correspondence and reprint requests to Dr. Craig J. Lobb, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39211. E-mail address: clobb@microbio.umsmed.edu 4 Abbreviations used in this paper: RSS, recombination signal sequence; CDR, complementarity determining region; cir-DNA, extrachromosomal circular DNA; FR, framework region; H, heavy chain of Ig; RAG, recombination activating gene.

required to provide combinatorial diversity. Second, DH segments contribute sequence diversity to the H chain complementarity-determining region 3 (CDR3) region. Lastly, the expression of the rearranged DJ segment may provide B cell regulatory function by preventing utilization of H chains with D regions in alternate reading frames. These regulatory mechanisms, known to occur in the mouse (5) but not in humans (6), are dependent upon the presence of initiation codons found upstream of murine DH segments. Phylogenetic studies have shown that the structure of VH and JH segments is conserved in lower vertebrates, but information on the structure, organization, and function of DH segments is limited. In the chicken, the H chain locus contains multiple VH gene segments, only 1 of which is functional, a single JH gene segment, and 16 DH gene segments (79). Fifteen of the DH segments are highly homologous suggesting that these arose by duplication. The D segments lack upstream initiation codons, are anked on both sides by RSS elements containing a 12-bp spacer, and P-, but not N-nucleotides, are observed at the DJ and VDJ junctions (9). In Xenopus, the only amphibian presently examined, only one complete sequence of a DH segment is known, but cDNA evidence indicates that other DH segments are likely present. This element is anked on both sides by RSS elements with 12-bp spacers, and the potential coding region is 6 nt in length. cDNA studies suggest that DH segments are a more important source of diversity in adults rather than tadpoles (10, 11). In the horned shark, H chain gene segments are closely linked within multiple gene clusters (12). The general organizational pattern of the segments within these clusters is V-D1-D2-J-C. The VH and JH segments have 23-bp RSS spacers located downstream and upstream, respectively, of their coding sequences. In 99% of these clusters, the D1 segment is anked by 12- and 23-bp RSS spacers, whereas the D2 segment is anked on both sides by 12-bp RSS spacers. There are 200 or more different gene clusters, and in about half of these clusters germline-joined VD or VDJ segments
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The Journal of Immunology exist. In the other clusters, recombination appears restricted to segments within a cluster and both the D1 and D2 segments appear to be utilized (13). The sequences of the D1 and D2 segments in different clusters are highly conserved which suggests that shark DH gene segments encode only limited CDR3 structural diversity. Studies with the channel catsh have provided insight into the early evolutionary patterns of Ig gene organization and genetic diversity. The genomic organization of H chain gene segments in the catsh, a teleost (bony) sh, is different from that known in sharks. The C gene, which encodes the predominant serum Ig and Ab of catsh (14, 15), exists as a single genomic copy (16, 17), a general conclusion that has been extended to other teleost sh (18 21). In addition, it has been shown that VH gene families extensively diverged at the level of the bony sh. Southern blot studies indicate an extensive genomic VH repertoire consisting of at least seven catsh VH gene families, which represent 120 different gene segments (22). Members of these different families are interspersed with one another in the locus and are closely linked. Each of the sequenced VH members has a typical RSS with a 22- to 24-bp spacer located downstream of the coding region (23). Studies on the JH locus of the catsh also indicate structural and organizational patterns similar to those found in higher vertebrates. Nine JH gene segments (designated JH1 through JH9) are tightly clustered within a 2.2-kb region located immediately upstream from C . Each JH segment appears to be functional, and each contains an RSS element with a 22- to 24-bp spacer located immediately upstream of the coding region (24, 25). These studies indicate that catsh VH and JH gene segments would not be expected to undergo VJ joining without violating the 12/23 rule of recombination (3). In addition, earlier cDNA analyses revealed sequence diversity within the H chain CDR3 region that was not encoded by VH or JH segments suggesting, that DH segments must contribute to CDR3 diversity in the catsh (26). However, at this point DH segments have not been identied in bony sh. With these studies showing that the structure and organization of VH and JH gene segments co-evolved with single-copy C region genes, it was important to determine whether DH segments are present, and if so to determine their structure and genomic organization. This report characterizes DH segments of the channel catsh and provides new insights into the early evolutionary patterns of Ig gene organization and the mechanisms of CDR3 diversity. Cloning and characterization of DH gene segments

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A previously constructed DASH II genomic library (24) was screened by hybridization using fragments obtained from the cir-DNA clones, and nine overlapping genomic clones were isolated. Southern blot analysis determined which EcoRI fragment contained DH gene segments, and these were subcloned into pBluescript SK( ). Overlapping nested deletion subclones were constructed using exonuclease III as previously described (25) and sequenced using Sequenase 2.0 (United States Biochemical).

RNA isolation, cDNA construction, and PCR approaches

Total RNA was puried by lysing PBL obtained from two adult channel catsh as described earlier (28). mRNA was puried from the total by oligo(dT) column elution (Qiagen, Chatsworth, CA) and double-stranded cDNA was synthesized utilizing Moloney murine leukemia virus (MMuLV) reverse transcriptase and oligo(dT) priming (Pharmacia Biotech, Piscataway, NJ). The V regions of the expressed Ig H chains were amplied by PCR using forward primers specic for the FR1 regions of the VH1VH6 variable gene families and a reverse primer specic for the C 1 domain. The sequences of the primers was as follows: VH1-FR1, 5 -ATGGACAGTC CCTGACC-3 ; VH2-FR1, 5 -G/TGAACTGACTCAGCCT-3 ; VH3-FR1, 5 -TATTCCTGCAGTCAGAC-3 ; VH4-FR1, 5 -GGGATGTGCAGTA GAAC-3 ; VH5-FR1, 5 -CTGAGCTCATCCAGCCA-3 ; VH6-FR1, 5 GCTGCTGGCAGCCGTAC-3 ; and C 118, 5 -GCCGCACTGCCA CACGGG-3 . Thirty cycles of PCR amplication were conducted using the GeneAmp DNA Amplication kit (Perkin-Elmer Cetus, Norwalk, CT), a Twin Block System thermocycler (Ericomp, San Diego, CA), and the following amplication parameters: 1 min at 94C, 2 min at 50C, and 3 min at 72C for a total of 30 cycles. The amplicons were puried and ligated into a T/A plasmid (Invitrogen; Carlsbad, CA). Clones were randomly chosen for sequencing. Database comparisons of the derived nucleic acid sequences were conducted with BLAST algorithm (29). Selected sequences were also analyzed using the Pustell DNA/Protein analysis program (IBI, New Haven, CT). Assignment of nucleotides to the framework region (FR) and CDR-encoded regions is according to Kabat et al. (30).

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Southern blot analysis

Genomic DNA, obtained from the nucleated erythrocytes of an individual adult channel catsh, was restricted and blotted onto a nylon membrane. The blots were hybridized with either a 700-bp StuI-StyI restriction fragment derived from a region located downstream of the DH1 germline gene segment or with a 950-bp SstI fragment derived from a region located downstream of the DH2 germline gene segment. The methods for labeling of genomic restriction fragments as well as conditions for Southern blot hybridization were identical to those described earlier (24).

Results
Characterization of DH-JH recombination signal joints in cirDNA of the channel catsh Inspection of H chain sequences derived from earlier cDNA studies indicated that it would be difcult to design specic primers for use in PCR strategies to characterize catsh germline DH segments. However, the catsh JH locus had been sequenced, and we reasoned that it should be possible to make a library derived from cir-DNA and effectively probe this library to identify possible excision products of DH to JH recombination events. A library was made using cir-DNA derived from lymphocytes of the anterior kidneys (a major hematopoietic organ in bony sh) of juvenile catsh using a modied alkaline lysis procedure with subsequent ATP-dependent DNase treatment. Replicate lifts of the library were screened with two different probes. The rst probe, a 1.5-kb EcoRI-ClaI fragment, begins immediately upstream of the JH locus and extends downstream to include the JH1 segment. The second probe, a 4.2-kb XbaI fragment, begins downstream of the JH locus and extends into the intron located between the C 2 and C 3 domains. Clones that hybridized under stringent conditions with the EcoRI-ClaI probe, and negatively with the XbaI probe, were considered as candidates representing the excision products of DH to JH rearrangement events.

Materials and Methods

Construction and analysis of channel catsh extrachromosomal circular DNA (cir-DNA) library
Cir-DNA was isolated from the anterior kidney of 30 individual 4- to 6-mo-old channel catsh, Ictalurus punctatus ( 26 g each) using a modied alkaline lysis procedure (27) with subsequent ATP-dependent DNase treatment (United States Biochemical, Cleveland, OH). Briey, 108 anterior kidney lymphocytes were lysed in a buffer containing 50 mM NaCl, 2 mM EDTA, and 1% SDS (pH 12.5). The solution was neutralized with the addition of 0.2 volumes of 1 M Tris-HCl (pH 7.0) and RNase treated. After the addition of 0.1 volumes of 5 M NaCl, the solution was proteinase K treated, phenol/chloroform extracted, and precipitated with 2 volumes of ethanol. The solution was then treated at 37C with 2 U/microgram of ATP-dependent DNase in a buffer containing 6.7 mM glycine (pH 9.4), 30 mM MgCl2, 8.3 mM 2-ME, 0.5 mM ATP, and 10 g/ml of BSA. A library was constructed from the enriched cir-DNA by cloning into the EcoRI site of ZAP II (Stratagene, La Jolla, CA). The library contained 2.3 105 recombinants and was subjected to one round of amplication. Replicate lifts were screened with two different probes. The rst, a 1.5-kb EcoRI-ClaI restriction fragment, represented a region immediately 5 of the JH locus. The second, a 4.2-kb XbaI restriction fragment, represented a region downstream of the JH locus and included the C 1 and C 2 coding region domains. Selected cir-DNA clones were subcloned into pBluescript SK( ).

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DH SEGMENTS OF THE CHANNEL CATFISH

FIGURE 1. Nucleotide sequences of the recombination signal joint identied in three cir-DNA clones aligned with the sequence of the germline JH and DH segments that were utilized in the recombination event. Clones Cir-E5, Cir-B1, and Cir-D2 were derived from cir-DNA obtained from anterior kidney lymphocytes of channel catsh and cloned in libraries. The nonamer (9-mer) and heptamer (7-mer) sequences of the RSS in JH and DH segments are boxed. Nucleotide identities with the cir-DNA sequence are indicated by dots. The nonamer and heptamer sequences in the 5 RSS of the DH segments are underlined.

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Representative clones that met the above criteria were sequenced. The partial nucleotide sequence for three of these cirDNA clones is shown in Fig. 1. A signal joint consisting of headto-head RSS elements was present in each clone. The signal joints consisted of a conserved nonamer, a 23-bp spacer, and heptamer immediately adjacent (or separated by one or two nucleotides) to a heptamer, 12-bp spacer, and a nonamer. The RSS elements in clones Cir-E5, Cir-B1, and Cir-D2 contained 23-bp spacers that were identical to the RSS elements of the germline JH8, JH3, and JH1 gene segments, respectively. Nucleotide identity with the 5 anking region of the respective JH segment continued further upstream (data not shown), and in each clone, the JH coding and 3 -anking regions were absent. This indicated that each clone represents an extrachromosomal product of a recombination event between a germline JH gene segment and a putative DH gene segment with an RSS element containing a 12-bp spacer. Initial sequence comparisons of clones Cir-E5 and Cir-B1 indicated that a similar DH segment was used in both rearrangements. The sequences of Cir-E5 and Cir-B1 were extended an additional 2 kb downstream of the DH RSS and only 8-bp differences were identied (data not shown). These analyses suggest that different alleles of the same DH segment were involved in these recombination events or that a family of DH segments whose anking regions are highly conserved exists. The DH segment utilized in Cir-D2 was different from that found in the other two clones. The sequence of clone Cir-D2 was extended about 500 bp into the DH anking region, and there was no signicant homology with the DH anking regions found in Cir-E5 or Cir-B1 (data not shown). The signal joint identied in clone Cir-B1 was precise. However, nucleotide insertions were observed between the abutted signal heptamers in the other two clones; Cir-E5 had a 1-bp insertion and Cir-D2 had a 2-bp insertion. These insertions are likely N-region additions to the DH-JH signal joint (see Discussion). Mapping, genomic organization, and structure of catsh germline DH gene segments To locate the germline DH gene segments utilized in these cirDNA clones, a channel catsh genomic library was initially screened with a 700-bp StuI-StyI probe derived from the DH anking region of Cir-E5 (designated 3 -DH1). Restriction mapping and hybridization analysis showed that four genomic clones (13-4, 136.1, 2-2, and 13-7) overlapped each other and overlapped a pre-

viously isolated clone, C7 (25), which contained the JH locus and the C (Fig. 2). The cumulative distance spanned by these overlapping clones is 30 kb. Each clone hybridized with the 3 -DH1 probe, but only clones 2-2 and 13-7 hybridized with a 4.1-kb BamHI-EcoRI probe derived from the DH anking region of clone Cir-D2 (designated 3 -DH2). It was concluded that the 4.3-kb and the 6.3-kb EcoRI fragments contained the regions that hybridized with the 3 -DH1 and the 3 -DH2 probes, respectively. The 4.3-kb EcoRI fragment was partially sequenced and a single germline DH gene segment, designated DH1, was identied. The 3 -RSS DH1 sequence was identical to the 3 -DH-RSS sequence found in clones Cir-E5 and Cir-D2 (Fig. 1). Restriction sites within the 3 -DH2 anking region placed the DH2 segment in a 2.2-kb EcoRI-XbaI fragment located at the 5 end of the 6.3-kb EcoRI fragment. This fragment was sequenced, and two DH segments were identied. The DH2 segment contained the identical DH-3 -RSS sequence that was identied in Cir-D2 (Fig. 1). Further upstream ( 0.8 kb) another DH segment, designated DH3, was identied whose sequence was distinct from DH1 and DH2. The nucleotide sequence of the three germline DH gene segments is shown in Fig. 3A. Each DH gene segment has a 5 - and 3 -RSS with a 12-bp spacer. All three DH gene segments are in the same transcriptional orientation relative to each other as well as to the downstream JH segments. The DH1 and DH2 gene segments have conserved heptamers (CACT/AGTG) 5 and 3 of the coding region; however, the 5 and 3 heptamers of the DH3 gene segment differ from the consensus by 3 bp and 2 bp, respectively. The 5 nonamer of each DH gene segment is T-rich, whereas the 3 nonamers are A-rich. In comparison to the DH1 gene segment, DH2 and DH3 have 8- and 7-bp identities in the 5 RSS spacer regions respectively, whereas the 3 RSS elements of DH2 and DH3 have 4- and 3-bp identities. Sequence comparisons showed that there was little, if any, sequence similarity in the 5 and 3 anking regions of the three DH segments (Fig. 3B). The potential coding regions of the DH segments are 1119 bp in length. Corbett et al. (6) in their study on human DH segments proposed that coding regions of DH segments could be characterized by the coding character of their amino acids rather than by dening the reading frames relative to the RSS as done by Ichihara et al. (31). Inspection indicates that catsh DH coding regions could be grouped into this same character pattern: one reading frame generally encodes polar/hydrophilic amino acids, a second

The Journal of Immunology

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FIGURE 2. Partial restriction map and genomic location of DH, JH, and C gene segments in the channel catsh. The restriction enzymes used to map the locus are indicated by single letter designations: E, EcoRI; H, HindIII; B, BamHI; S, SstI; and Bs, BstEII. The location of the four C domains (stippled boxes) and the JH and DH gene segments (solid boxes) are indicated. The positions of the overlapping genomic clones (13-7, 2-2, 13-6.1, 13-4, and C7) that span the locus are shown beneath the restriction map. The sizes of the internal EcoRI restriction fragments within these clones are shown in italics. The stippled bars shown above the restriction map indicate the relative locations of the 950-bp SstI and the 700-bp StuI-StyI restriction fragments that were used in Southern blot analyses.

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generally encodes hydrophobic amino acids, and a third features a stop codon(s) (Fig. 3A). Genomic Southern analyses indicate that catsh DH1 and DH2 represent single member gene families The germline DH gene segments in murine and human H chain loci are grouped as families as initially demonstrated by genomic hybridization experiments (32, 33). Subsequent sequence analysis showed that members of the same family share nucleotide homology in the coding regions, RSS elements, and anking regions (6). To determine whether families of DH gene segments are present in the catsh, probes were derived from the 3 anking regions of the DH1 and the DH2 segments and used in genomic Southern blots. If multiple DH family members exist in catsh, then Southern blot analysis should reveal the presence of different hybridizing fragments. However, these studies showed that only a single band (or

two bands which were readily interpreted by the genomic map and sequence) were dened when the Southern blots were hybridized with these probes (Fig. 4). Expression of DH13 segments and analysis of the CDR3 region of catsh H chain cDNA Based on genomic sequence data, each of the three DH gene segments appears functional. The functionality of these segments would be supported if each could be identied in an expressed VDJ rearrangement. Toward this end, cDNA was constructed from mRNA obtained from the PBL of two adult channel catsh. Following cDNA synthesis, forward primers corresponding to the FR1 region of six different catsh VH families were used in conjunction with a reverse primer for the C 1 in PCR studies. The PCR products were cloned, and representative clones from these families were sequenced. Members of the VH7 family were not

FIGURE 3. Nucleotide sequence comparison of the catsh DH1, DH2, and DH3 segments. A, The nonamer (9-mer) and heptamer (7-mer) sequences of the 5 and 3 RSS elements are boxed. The predicted amino acids encoded by the three reading frames within the DH coding region are indicated. The reading frames that principally encode hydrophilic/polar, hydrophobic, or stop codon(s) are indicated. B, Partial sequence of the immediate 5 and 3 anking regions of the DH1, DH2, and DH3 segments. Only the nonamer sequences within the DH 5 and 3 RSS are shown.

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DH SEGMENTS OF THE CHANNEL CATFISH ed that utilized the full-length DH1, DH2, or DH3 coding region. Nucleotide deletions from the DH coding regions were found at the 5 and the 3 ends, or from both the 5 and 3 ends. P-nucleotide additions were also identied in sequences that utilized the fulllength 5 or 3 ends of the DH coding region. Most of the P-region additions, one or two nucleotides in length, were located at the 3 end of the DH coding region, although one sequence (5.rh8) had a P-nucleotide added to the 5 end of the DH3 coding region. The expressed reading frame of the DH segments also exhibited variation. Examples of sequences that utilized the polar/hydrophilic, hydrophobic, or stop DH1 reading frame are shown in Table I. The stop reading frame of DH2, as well as the hydrophobic reading frame of DH3 was not utilized in any of the cDNA clones that were sequenced. JH-encoded sequences were readily assigned to segments JH1 JH9, except for clone 6.rh11 that may have used JH3, JH4, or JH5. In all but two of these sequences, nucleotides were deleted from the 5 end of the JH coding region. The number of bases deleted from these JH segments averaged 4.4 and ranged in length from 37 bases. In two sequences that expressed the full-length JH coding region (1.rh17 and 4.rh18), P-region additions of 4 and 5 nt, respectively, were added at the 5 end of the JH coding region. Based on limited information on catsh VH germline sequences, the CDR3-encoded region of germline VH segments representing these different families is generally 4 nt in length (Ref. 23 and our unpublished data). Nucleotides that are identical to those present within characterized germline members of these six VH families are indicated in Table I. These analyses indicate that nucleotides can be deleted from the germline CDR3 encoded VH region. In one example, in clone 3.rh14 where the full-length VH region was likely expressed, a single P-region nucleotide was represented. These analyses also provided information on nontemplated or N-region additions. N-region additions were located between V-D junctions as well as between D-J junctions. Because it is difcult to strictly assign N-region nucleotides to VD junctions, more informative data were derived from the D-J junctions. N-region additions in the DJ junctions of these clones averaged 4.7 bp and ranged in length from 1 to 11 bp. The majority of these nucleotides

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FIGURE 4. Southern blots of restricted genomic DNA hybridized with DH anking region probes. The DNA was restricted with the following enzymes: E, EcoRI; H, HindIII; B, BstEII; P, PstI; S, SstI; or X, XbaI. A, The restricted DNA was hybridized with a 700-bp StuI-StyI fragment derived from the 3 anking region of the catsh DH1 segment. B, The restricted DNA was hybridized with a 950-bp SstI restriction fragment derived from the 3 anking region of the catsh DH2 segment.

analyzed because it is a small family representing less than 10 members (22). The encoded V region was compared with germline sequences, and the utilized coding regions of the VH, DH, and JH segments were assigned. Representative sequences that used longer DH encoded regions and provided information regarding the mechanisms of VDJ joining are presented in Table I. The sequences that utilized the DH1, DH2, or DH3 segments showed that there was extensive coding-end processing consisting of both the removal and addition of various numbers of nucleotides from the VH, DH, and JH segments. Only one cDNA sequence was identi-

Table I. Assignment of nucleotides within the CDR3 encoded region of different channel catsh Ig heavy chain cDNA clones to germline VH, DH, and JH segments
Clonea Germline 6.rh11 3.rh14 3.rh12 3.rh11 1.rh6 1.rh16 Germline 3.rh13 1.rh17 2.rh12 6.rh14 Germline 5.rh8 4.rh18 1.rh12 V/Nb DH 1 GAAAGG GACCGf/AGAG GGG CGTCAC TTCGT CCACCGGG DH2 GCCC A GGGT TC DH3 AGAGGT TACGTGGA GGAGAGGGGCGC PDc D GTTATAGCAGCTGGGGTAG GTTATAGCAGtTGGGGTAG TATAGCAGCTGGGGTAG TATAGCAGtTGGGGTAG AGCAGCTGGGGTA CAGCTGGGGTAG GCTGGGGTgG PDc C CT C C N CGGA CCCCT ACGGGAGCACT A GTCCT TCC PJc J TTTGACTAC ATGCTTTTGACTAC CTACTTTGACTAC CTTTGCCTAC CAGCTACTTCGACTAC GCTTTTGATgAC JH# *e 8 3 1 2 7 8 7 6 4 8 9 9 DHRFd

CAATATAGCGGGT CAATATccCGGGT AC AATATAGC ATATAGCGGG TAGCGGG ATAACTACGGC ATAACTACGGC AcAACTA CTACGGC GC

GCCGTCTTC TCGT CCAGGG CCTCCC

GCTTTTGACTAC ACGATGCcTTTGATTAC CTACGACTACTTTGACTAC TACTTTGACTAC

CTTTT TCG TC

AGTTA

TGCTTTTGACTAC TAACTGGGCTTTTGATTAC CTGGGCTTTTGATTAC

s s

a Clones are designated by VH family with a period separating the cDNA clone number. Nonfunctional transcripts are underlined and have the following characteristics: 3.rh13, frameshift within the N-region addition downstream of DH2; and 1.rh12 has a stop codon within the VD junction. b Underlined nucleotides indicate identity with nucleotides in the CDR3 encoded region of characterized VH germline genes. N refers to nontemplated nucleotide additions located between segments. c Refers to P (palindromic) nucleotide additions located at the boundaries of the full-length ends of D and J junctions, respectively. d , , and s refer to the hydrophilic/polar, hydrophobic, and stop codon DH reading frames, respectively (see Fig. 3). e JH3, JH4, or JH5 could have been utilized in this cDNA clone. All other clones are assigned to the specic JH segment that was utilized (JH1 through JH9; Ref. 25). f This G nucleotide is likely a P-nucleotide addition from a full-length VH2 germline gene.

The Journal of Immunology were pyrimidines, principally C. Base stacking of pyrimidine or purine nucleotides was a common feature of these N-region additions. There did not appear to be a strong correlation between the length of the DH- and JH-encoded regions and the number of Nnucleotides between these junctions. There was also evidence of point mutations within the utilized DH and JH segments. Whether these represent somatic or perhaps allelic differences will have to await additional studies. Thus cDNA analyses indicate that DH1, DH2, and DH3 are functional DH segments that can undergo productive VDJ rearrangements.

1921 about 7.6 kb and, as shown by genomic Southern blot studies, the DH1 and DH2 segments represent single member families. Thus unlike the DH segments of mouse and humans that can be grouped into families based upon similarities in their coding as well as anking region sequences (6, 32, 33), the anking regions of catsh DH segments are apparently unrelated. These results suggest that these catsh DH segments did not evolve through recent gene duplication events. These results also suggest a phylogenetic primitiveness, and importantly, indicate that evolutionary pressures exerted during early phylogeny may have been conned to the sequence of the DH gene segment itself (the RSS, spacer, and coding region) and not upon potential sequences located within the immediate anking regions. The coding regions of catsh DH1, DH2, and DH3 segments (19, 13, and 11 bp in length, respectively) are shorter than many of the DH segments known in mammals. In humans, for example, there are seven DH families and the segments within a family generally exhibit a similar range in the length of their coding region. These coding regions range in size from the smallest family (DHQ52) that is 11 bp to the DH4 family, which is 3137 bp in length (6). In each of these DH families, one reading frame encodes one or more stop codons, a second tends to encode glycine residues in conjunction with polar/hydrophilic residues, and a third is hydrophobic in character. Corbett et al. (6) used this approach to classify reading frame usage rather than referencing the reading frame relative to the RSS. Reynaud et al. (7) in their earlier study had observed this DH coding character with the reading frames of avian DH segments. The reading frames of the catsh DH segments were readily assigned by this classication approach with similar amino acids represented. This pattern of reading frames is not apparent in shark DH segments. In the horned shark (Heterodontus) there are 200 genomic gene clusters, and in about half of these clusters there is germline joining of V-D or V-D-J. In the other half of these clusters the DH1 and DH2 segments, although distinct from one another, appear to vary by no more than a single nucleotide when compared with homologous segments in other clusters. Recombination events appear to be restricted to segments within a cluster, and both D1 and D2 appear to be utilized even though the 12/23 rule would allow only the D2 segment to be selected (12, 13). None of the shark DH sequences we examined had a reading frame that encoded a stop codon. The reading frames generally encode both hydrophobic as well as hydrophilic/polar amino acids. If, as suggested, an early role for DH gene segments is a medium for junctional diversity and somatic mutation (13), these studies indicate that bony sh are the rst vertebrates to evolve structurally distinct DH gene segments. DH1 is the only one of these gene segments that contains a start codon and a contiguous open reading frame that extends through the coding region. This start codon is located within the spacer region of the 5 RSS, and A/T rich regions are located upstream that may serve as promoter and transcription initiation sites. Transcriptional enhancer elements have been mapped 3 of the TM2 exon of catsh gene (41); hence a DH1-J recombination product might be transcribed and translated. It seems, however, that if a mechanism existed to regulate DH reading frame usage, similar to the well-characterized counter-selection mechanism of the D protein in murine systems (5), each of the catsh DH elements would share these structural features. This is not the case. In addition, the DH1 segment is used in all three reading frames (Table I), which also suggests that a mechanism to regulate DH reading frame usage by the production of a D protein may not exist. The genomic clones that contained the DH segments were hybridized with family-specic VH probes and none of the probes

Discussion
These results allow several conclusions to be made regarding the structure, genomic organization, and diversity patterns of Ig DH segments in early vertebrate phylogeny. The analysis of cir-DNA derived from anterior kidney lymphocytes of juvenile channel catsh dened the excision products of D-J recombination events. The sequenced clones contained recombination signal joints represented by head to head joining of DH and JH RSS elements. Sequence comparisons indicated that the anking as well as the RSS elements with 23-bp spacers were identical to previously characterized germline JH gene segments. The RSS elements with 12-bp spacer were identical to subsequently characterized DH segments. Hybridization studies indicated that the DH1 and DH2 segments were represented in the cir-DNA library in approximately equal numbers. A cir-DNA library, derived from anterior kidney lymphocytes of two adult catsh, was also constructed, and no positive clones were identied using the identical screening approach even though this library was nearly twice as large. These data suggest that the frequency of DH to JH recombination is higher in anterior kidney lymphocytes of juvenile rather than adult catsh, although it also possible that deletion circles from adult sh may more difcult to isolate if additional DH segments reside further upstream. Nonetheless, these results establish that the gene recombination mechanisms that result in the excision of DH-JH signal joints evolved early in vertebrate phylogeny. The signal joints of clones Cir-E5 and Cir-D2 had nucleotide insertions between the DH and JH RSS. During excision of the RSS, recombination activating gene-1 (RAG-1) and RAG-2 have been shown to associate with all four ends of the mammalian recombination products. Coding joint processing appears to result in a hairpin structure, whereas processing of the signal joints results in an open-ended or blunt-end structure (reviewed in Refs. 4 and 34). Because RAG and TdT are present in sh (3538), the additional nucleotides between the DH and JH RSS are probably Nregion additions attributable to TdT following RAG-mediated cleavage. The analyses of mammalian recombination signal joints indicate that N-region additions are found in high frequency in TCR recombination events (39), but less commonly observed in Ig recombination events. Murine pre-B cell transfection studies with recombination substrates indicated that N-region additions are present in an average of 14% of the signal joints formed. These nucleotides are mostly G and C and their presence correlated with TdT activity (40). Sequence comparison of the DH1 and DH2 anking regions in the cir-DNA clones indicated little structural similarity. As a result, DH1- and DH2-specic probes were derived and used to isolate and characterize a set of overlapping genomic clones. Based upon mapping and sequencing studies, the DH1 segment is JH proximal and located about 8.8 kb upstream from the JH1 segment. The DH2 segment is located about 6.8 kb upstream from DH1. The DH3 segment, which was identied by sequencing, is located about 0.8 kb upstream from DH2. These DH segments span a region of

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1922

DH SEGMENTS OF THE CHANNEL CATFISH

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FIGURE 5. Nucleotide sequence comparison of catsh DH13 segments with DH segments of other vertebrates. A, The sequences of the DHQ52 segments identied in mammals of different lineages are aligned with DH segments from the catsh, Xenopus (10), and shark (12). Conserved sequence motifs within these segments are underlined. B, The sequence of the human DH2-2 segment (shown double-stranded, Ref. 6) aligned with the catsh DH1 segment. The sequence of catsh DH1 segment is shown in the 3 -5 direction. Identical nucleotides in both sequences are shown in capital letters. The conserved coding region sequence located in both human DH2-2 and catsh DH1 is underlined.

hybridized under relaxed or stringent conditions (data not shown). This indicates that VH segments do not appear to be present within the examined region of the catsh DH locus. An earlier study with the coelacanth Latemeria had suggested that VH and DH segments were interspersed; putative DH segments were located immediately downstream of the VH gene segments (42). Although this study did not show that the gene segments were utilized nor were the location of JH segments identied, this study did suggest an alternate pattern of Ig V region gene organization. The distance separating the DH and JH loci of humans and mice (excluding the DHQ52 segment) is 20 kb (6, 43, 44). The relatively short distance separating the DH and JH loci in channel catsh parallels the relatively short distance separating the JH locus and the C (1.8 kb). In addition, earlier results showed that members of the different VH families are closely linked (average distance between segments of about 3 kb) and interspersed with each other (23). Pulsed eld studies have also determined that catsh VH segments are linked to JH and C on the same large genomic fragments (T. VenturaHolman and C. J. Lobb, manuscript in preparation). It appears that the structure and characteristic organizational pattern of Ig H chain V region gene segments of higher vertebrates evolved in a compact locus early in vertebrate phylogeny. Bony sh, as represented by studies in the channel catsh, appear to have been the earliest vertebrates to evolve multiple VH gene families upstream of different DH gene segments located in a dened regional arrangement and closely linked to the JH locus. The analysis of H chain cDNA to dene the junctional mechanisms of CDR3 diversity showed that there is extensive processing of the coding ends of the catsh VH, DH, and JH segments during recombination. Representative cDNA sequences showed that the DH1DH3 segments are functional and that these segments are used in different reading frames. Although the full-length coding region of the DH segments was expressed in some cDNA clones, deletion of nucleotides from the 5 end, the 3 end, or both ends of the DH coding region was generally observed. Deletion of nucleotides from the CDR3 encoded region of germline VH and JH

coding region was also observed. Of particular importance in these studies is that there is further junctional modication of the coding region ends by the addition of N- and P-region nucleotides. These analyses are the rst studies in bony sh that have the necessary germline information to indicate these processes. These patterns parallel those known from mammalian studies and imply that the mechanisms that provide for junctional diversity of Ig V region genes evolved early in phylogeny and are present at the level of the bony sh. Approximately 50% of the 50 H chain cDNA clones analyzed in this study utilized DH1, DH2, or DH3 in CDR3. The DH segments utilized in 25% of the remaining clones could not be determined because the length of the CDR3 was generally less than 8 nt when VH and JH contributed nucleotides were identied, and the majority of these nucleotides appeared to represent N-region additions. In the other 25% of the clones, sequence comparisons of the CDR3 indicate that there may be one additional germline DH segment represented among these sequences. Thus it is possible that there are a few other, as yet uncharacterized germline DH sequences, but present cDNA analyses do not indicate an extensive repertoire of germline DH segments in the catsh. Only about 5% of these cDNA sequences utilized DH3 and inspection of the germline DH3 sequence indicates that the RSS heptamers sequences vary, although the consensus nonamers are conserved. Changes from consensus in the three heptamer nucleotides adjacent to the coding region have been shown to be the most deleterious for recombination (34). Although these heptamer nucleotides are conserved in each of the three characterized DH segments (Fig. 3), changes in other positions are known to effect recombination frequencies in mammalian systems and may account for the decreased usage of catsh DH3 in these analyses. Inspection of the catsh DH segments indicates conservation of internal coding region sequence motifs. This led us to determine whether any of the sequence motifs present in the coding region of the catsh DH segments were conserved in the DH segments of

The Journal of Immunology other vertebrates. Comparative studies of the DH segments in humans, mice, rabbits, and the primitive insectivore, Suncus murinus, have shown that the DHQ52 segment is conserved in both its structure and its location within mammalian IgH loci (45 48). It represents a single member gene family and is the only DH segment conserved in these diverse lineages of mammals. In each species, the DHQ52 segment is JH proximal and located less than 1 kb from the JH region. These studies suggest that this DH segment is monophyletic and that it may have emerged before the radiation of mammals. The multiple alignment of the DHQ52 segment in these species indicates that the 10 11 bp coding sequence is conserved around the core internal sequence TAACTGGG (Fig. 5A). The alignment of catsh DH13 indicates that the internal coding motifs TAACT and CTGGG are represented in the catsh DH segments as well as in shark DH segments. The DH1 and DH2 coding regions also share the common internal sequence TATAGC. The sequence databases were searched with the nucleotide sequences of the catsh DH1DH3 segments using the BLAST algorithm (29). These analyses showed that the sequence of the catsh DH1 was conserved in DH segments of other vertebrates. The alignment with the DH2-2 segment of humans is shown in Fig. 5B. As indicated, the catsh DH1 coding region is identical in 13 of the 14 positions with the reverse complement of the DH2-2 coding strand. Although this phylogenetic relationship might indicate conserved Ag recognition motifs, its importance may be in its relationship with the binding of enzymes within the recombinase complex. Notable conservation of sequence motifs also occurs within the coding and the RSS regions of catsh VH, DH, and JH segments. In catsh VH segments the conserved FR3 sequence 5 -GTGTAT TACTGTG partially encodes the YYC sequence, a structural hallmark of VH genes, and contains an embedded heptamer, which in mammalian systems is known to be involved in secondary VH recombination events (49, 50). This VH consensus sequence is conserved within the 5 RSS of catsh DH segments. The terminal nine nucleotides of the VH consensus sequence are repeated downstream of the RSS of the catsh JH segments where the sequence 5 -AYTACTGGG (where Y is T or C) is located. This sequence contains the codon for the FR4 tryptophan that is a structural hallmark of JH segments. There is considerable evidence, much of it attained from experiments with extrachromosomal recombination substrates, which show that anking nucleotides within the coding region inuence recombination frequencies. Gerstein and Lieber (51) demonstrated that terminal coding region nucleotides adjacent to the heptamer inuenced recombination efciencies over at least a 260-fold range. A or T nucleotides adjacent to the heptamer reduce recombination frequencies, but these effects are mediated by nucleotides in coding positions 6 8 nt away from the RSS (52, 53). It is reasonable to postulate that these phylogenetically conserved sequence motifs might serve additional functions perhaps related to the association of the recombinase complex. These phylogenetically conserved VH, DH, and JH sequence motifs might also reect recognition sites for primitive rearranging gene segments. Recent studies have shown that there appears to be a common evolutionary origin for the RAG proteins and transposases suggesting that transposons played an active role in the evolution of the immune system (54, 55). We recently identied transposons of the Tc1/mariner family located within the H chain locus of the channel catsh and these studies also suggest that transposons may have contributed to the structure and organization of the catsh IgH locus (56). Sakano et al. (57) in their early studies of L chain gene recombination postulated that the heptamer and nonamer of V and J segments resembled the inverted repeated ends of transposons. The sequence of catsh DH segments phylogenetically supports this hypothesis. Alignment of the catsh

1923 DH segments shows that the 5 RSS and the 3 RSS can be considered as inverted terminal repeats that anks the DH coding region. Five to seven nucleotides within the spacer region of the different DH in addition to the heptamer and the nonamer form this structure. In conclusion, these studies have shown that the structure and genomic organization of DH segments in a bony sh parallels that known in higher vertebrates. Different DH segments, which contain 12-bp RSS on both the 5 and 3 ends with phylogenetically conserved heptamers, nonamers, and coding region sequence motifs, are located in a dened regional arrangement immediately upstream from the JH locus. Germline recombination of DH to JH segments can lead to the excision of extrachromosomal cir-DNA that contains the DH-JH recombination signal joint, and these signal joints may be modied by the addition of N-region nucleotides. cDNA studies indicate that the same DH segment can be expressed with different JH segments and members of different VH families and thus there is combinatorial diversity of H chain V region gene segments. These studies also have shown that there is extensive junctional modication of the VH, DH, and JH coding region ends during recombination. These modication processes include nucleotide deletion, N- and P-region nucleotide additions, and alternate DH reading frame usage. These combined studies indicate that the structure, genomic organization, and general patterns of DH gene recombination that are typically associated with higher vertebrates evolved early in phylogeny at the level of the bony sh.

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