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Heavy Chain Diversity Region Segments of
the Channel Catfish: Structure, Organization,
Expression and Phylogenetic Implications
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J Immunol 2000; 164:1916-1924; ;
doi: 10.4049/jimmunol.164.4.1916
http://www.jimmunol.org/content/164/4/1916
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References
J. Russell Hayman and Craig J. Lobb
Heavy Chain Diversity Region Segments of the Channel
Catfish: Structure, Organization, Expression and Phylogenetic
Implications1,2
J. Russell Hayman and Craig J. Lobb3
D
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
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
This work was supported by Grant AI23052 from the National Institutes of Health.
2
The sequences discussed in this paper have been entered into the GenBank database
under the accession numbers AF161271–AF161289.
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: [email protected]
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.
Copyright © 2000 by The American Association of Immunologists
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 (7–9). Fifteen of the DH segments are highly
homologous suggesting that these arose by duplication. The D segments lack upstream initiation codons, are flanked 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 flanked
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 flanked by 12- and 23-bp RSS
spacers, whereas the D2 segment is flanked 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
0022-1767/00/$02.00
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Circular DNA, derived from lymphocytes of juvenile channel catfish, 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 flanking regions within the clones were used to probe a genomic library. Three
germline DH gene segments containing 11–19 bp coding regions flanked by 12-bp RSS elements with conserved heptamers and
nonamers were identified. 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 catfish 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 fish. The Journal of Immunology, 2000, 164: 1916 –1924.
The Journal of Immunology
Materials and Methods
Construction and analysis of channel catfish extrachromosomal
circular DNA (cir-DNA) library
Cir-DNA was isolated from the anterior kidney of 30 individual 4- to
6-mo-old channel catfish, Ictalurus punctatus (⬃26 g each) using a modified alkaline lysis procedure (27) with subsequent ATP-dependent DNase
treatment (United States Biochemical, Cleveland, OH). Briefly, 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 37°C 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 amplification.
Replicate lifts were screened with two different probes. The first, 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(⫺).
Cloning and characterization of DH gene segments
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 purified by lysing PBL obtained from two adult channel
catfish as described earlier (28). mRNA was purified 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 amplified by PCR
using forward primers specific for the FR1 regions of the VH1–VH6 variable gene families and a reverse primer specific 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␮1–18, 5⬘-GCCGCACTGCCA
CACGGG-3⬘. Thirty cycles of PCR amplification were conducted using
the GeneAmp DNA Amplification kit (Perkin-Elmer Cetus, Norwalk, CT),
a Twin Block System thermocycler (Ericomp, San Diego, CA), and the
following amplification parameters: 1 min at 94°C, 2 min at 50°C, and 3
min at 72°C for a total of 30 cycles. The amplicons were purified 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).
Southern blot analysis
Genomic DNA, obtained from the nucleated erythrocytes of an individual
adult channel catfish, 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 catfish
Inspection of H chain sequences derived from earlier cDNA studies indicated that it would be difficult to design specific primers for
use in PCR strategies to characterize catfish germline DH segments. However, the catfish 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 fish) of juvenile
catfish using a modified alkaline lysis procedure with subsequent
ATP-dependent DNase treatment. Replicate lifts of the library
were screened with two different probes. The first 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.
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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 catfish 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 catfish, a teleost (bony) fish, is different from that known in
sharks. The C␮ gene, which encodes the predominant serum Ig
and Ab of catfish (14, 15), exists as a single genomic copy (16, 17),
a general conclusion that has been extended to other teleost fish
(18 –21). 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 catfish 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 catfish 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 catfish 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 catfish (26).
However, at this point DH segments have not been identified in
bony fish. 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 catfish and provides new insights into the early evolutionary patterns of Ig gene organization and the mechanisms of
CDR3 diversity.
1917
1918
DH SEGMENTS OF THE CHANNEL CATFISH
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⬘
flanking region of the respective JH segment continued further upstream (data not shown), and in each clone, the JH coding and
3⬘-flanking 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 identified (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 flanking 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
flanking region, and there was no significant homology with the
DH flanking regions found in Cir-E5 or Cir-B1 (data not shown).
The signal joint identified 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 catfish
germline DH gene segments
To locate the germline DH gene segments utilized in these cirDNA clones, a channel catfish ␭ genomic library was initially
screened with a 700-bp StuI-StyI probe derived from the DH flanking 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 flanking 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 identified. 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
flanking 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 identified.
The DH2 segment contained the identical DH-3⬘-RSS sequence
that was identified in Cir-D2 (Fig. 1). Further upstream (⬃0.8 kb)
another DH segment, designated DH3, was identified 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⬘
flanking regions of the three DH segments (Fig. 3B).
The potential coding regions of the DH segments are 11–19 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
defining the reading frames relative to the RSS as done by Ichihara
et al. (31). Inspection indicates that catfish DH coding regions
could be grouped into this same character pattern: one reading
frame generally encodes polar/hydrophilic amino acids, a second
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FIGURE 1. Nucleotide sequences of the recombination signal joint identified 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 catfish 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.
The Journal of Immunology
1919
FIGURE 2. Partial restriction map and genomic location of DH, JH, and C␮ gene segments in the channel catfish. 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.
Genomic Southern analyses indicate that catfish 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 flanking regions (6).
To determine whether families of DH gene segments are present in
the catfish, probes were derived from the 3⬘ flanking regions of the
DH1 and the DH2 segments and used in genomic Southern blots. If
multiple DH family members exist in catfish, 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 defined when the Southern blots were hybridized
with these probes (Fig. 4).
Expression of DH1–3 segments and analysis of the CDR3 region
of catfish 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 identified in an expressed
VDJ rearrangement. Toward this end, cDNA was constructed from
mRNA obtained from the PBL of two adult channel catfish. Following cDNA synthesis, forward primers corresponding to the
FR1 region of six different catfish 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 catfish 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⬘
flanking 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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generally encodes hydrophobic amino acids, and a third features a
stop codon(s) (Fig. 3A).
1920
DH SEGMENTS OF THE CHANNEL CATFISH
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 catfish Ig heavy chain cDNA clones to germline VH, DH, and
JH segments
Clonea
V/Nb
Germline
6.rh11
3.rh14
3.rh12
3.rh11
1.rh6
1.rh16
DH 1
GAAAGG
GACCGf/AGAG
GGG
CGTCAC
TTCGT
CCACCGGG
Germline
3.rh13
1.rh17
2.rh12
6.rh14
DH2
GCCC
A
GGGT
TC
Germline
5.rh8
4.rh18
1.rh12
DH3
AGAGGT
TACGTGGA
GGAGAGGGGCGC
PDc
D
GTTATAGCAGCTGGGGTAG
GTTATAGCAGtTGGGGTAG
TATAGCAGCTGGGGTAG
TATAGCAGtTGGGGTAG
AGCAGCTGGGGTA
CAGCTGGGGTAG
GCTGGGGTgG
PDc
C
CT
C
C
CAATATAGCGGGT
CAATATccCGGGT AC
AATATAGC
ATATAGCGGG
TAGCGGG
T
ATAACTACGGC
ATAACTACGGC
AcAACTA
CTACGGC GC
N
PJc
CGGA
CCCCT
ACGGGAGCACT
A
GTCCT
TCC
GCCGTCTTC
TCGT
CCAGGG
CCTCCC
CTTTT
TCG
TC
AGTTA
JH#
DHRFd
TTTGACTAC
ATGCTTTTGACTAC
CTACTTTGACTAC
CTTTGCCTAC
CAGCTACTTCGACTAC
GCTTTTGATgAC
*e
8
3
1
2
7
⫺
⫹
⫹
⫹
⫹
s
GCTTTTGACTAC
ACGATGCcTTTGATTAC
CTACGACTACTTTGACTAC
TACTTTGACTAC
8
7
6
4
⫺
⫹
⫺
⫹
TGCTTTTGACTAC
TAACTGGGCTTTTGATTAC
CTGGGCTTTTGATTAC
8
9
9
⫹
s
s
J
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 specific 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.
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FIGURE 4. Southern blots of restricted genomic DNA hybridized with
DH flanking 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⬘ flanking region of the catfish DH1 segment. B, The restricted DNA was hybridized with a 950-bp SstI restriction fragment derived from the 3⬘ flanking region of the catfish DH2 segment.
fied 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 identified 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
3–7 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 catfish 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 difficult
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
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.
Discussion
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
flanking region sequences (6, 32, 33), the flanking regions of catfish DH segments are apparently unrelated. These results suggest
that these catfish 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 confined to the sequence of the DH gene segment itself (the RSS, spacer, and coding
region) and not upon potential sequences located within the immediate flanking regions.
The coding regions of catfish 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 31–37 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 catfish DH segments were readily assigned by this classification 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 fish are the first 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 catfish ␮ 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 catfish 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-specific VH probes and none of the probes
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
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 catfish defined 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 flanking 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 catfish, was also constructed, and no
positive clones were identified 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 catfish,
although it also possible that deletion circles from adult fish may
more difficult 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 fish (35–38), 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 flanking regions in
the cir-DNA clones indicated little structural similarity. As a result, DH1- and DH2-specific 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 identified by sequencing, is located about
0.8 kb upstream from DH2. These DH segments span a region of
1921
1922
DH SEGMENTS OF THE CHANNEL CATFISH
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 catfish 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 identified, 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 catfish 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 field
studies have also determined that catfish 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 fish, as represented by
studies in the channel catfish, appear to have been the earliest
vertebrates to evolve multiple VH gene families upstream of different DH gene segments located in a defined regional arrangement
and closely linked to the JH locus.
The analysis of H chain cDNA to define the junctional mechanisms of CDR3 diversity showed that there is extensive processing
of the coding ends of the catfish VH, DH, and JH segments during
recombination. Representative cDNA sequences showed that the
DH1–DH3 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 modification of the coding
region ends by the addition of N- and P-region nucleotides. These
analyses are the first studies in bony fish 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 fish.
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 identified, 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 catfish.
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 catfish DH3 in these analyses.
Inspection of the catfish 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 catfish DH segments were conserved in the DH segments of
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FIGURE 5. Nucleotide sequence comparison of catfish DH1–3 segments with DH segments of other vertebrates. A, The sequences of the DHQ52
segments identified in mammals of different lineages are aligned with DH segments from the catfish, 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 catfish DH1
segment. The sequence of catfish 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 catfish DH1 is underlined.
The Journal of Immunology
DH segments shows that the 5⬘ RSS and the 3⬘ RSS can be considered as inverted terminal repeats that flanks 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 fish 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 defined 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 modified 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 modification of the VH, DH, and JH coding region ends
during recombination. These modification 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 fish.
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DH SEGMENTS OF THE CHANNEL CATFISH