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The Journal of Immunology, 2002, 169: 2488-2497.
Copyright © 2002 by The American Association of Immunologists

The IgH Locus of the Channel Catfish, Ictalurus punctatus, Contains Multiple Constant Region Gene Sequences: Different Genes Encode Heavy Chains of Membrane and Secreted IgD1

Eva Bengtén2,*, Sylvie M.-A. Quiniou3,*, Tor B. Stuge4,*, Takayuki Katagiri5,*, Norman W. Miller*, L. William Clem*, Gregory W. Warr{dagger} and Melanie Wilson*

* Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216; and {dagger} Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The {delta}-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to Cµ1, seven C domain-encoding exons ({delta}1–{delta}7), and a transmembrane tail. The presence of cDNA forms showing splicing of {delta}7 to an exon encoding a secretory tail was interpreted to indicate that membrane ({delta}m) and secreted ({delta}s) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three {delta} C region genes, each linked to a µ gene or pseudogene. The first (IGHD1) is located 1.6 kb 3' of the functional Cµ (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo Cµ (IGHM3P), ~725 kb 5' of IGHM1. These two {delta} genes are highly similar in sequence and each contains a tandem duplication of {delta}2-{delta}3-{delta}4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a µ pseudogene indicates that the putative {delta}s product may not be expressed as a chimeric µ{delta} molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish {delta}m transcripts appear to originate from IGHD1, whereas {delta}s transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed {delta} gene. The third {delta} (IGHD2) is associated with a pseudo Cµ (IGHM2P); its presence is inferred by Southern blot analyses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The predominant teleost serum Ig has been well characterized at both the protein and molecular levels and is considered to be a tetrameric homolog of IgM (reviewed in Refs. 1 and 2). In contrast, much less is known about the teleost homolog(s) of IgD. Before the description of a {delta} homolog in channel catfish, Ictalurus punctatus (3), IgD had been found only in primates and rodents (4, 5, 6, 7). The catfish {delta} homolog, initially identified at the cDNA level, was unique in that it was expressed as a chimeric molecule consisting of a rearranged VDJ, a Cµ1 exon, and seven novel C region domains, some of which showed sequence homology to mammalian {delta} by phylogenetic analyses. Full-length cDNAs for the membrane ({delta}m) form and partial cDNAs for the secreted ({delta}s) form of catfish {delta} were identified (3). In addition, these earlier studies demonstrated that the catfish {delta}1 exon was located 1.6 kb 3' of the µ gene and that identifiable class-switch sequences were absent from this short intron. These prior findings suggested that catfish {delta} message, like that of mammalian {delta}, is produced by alternative mRNA splicing rather than class switching involving chromosomal recombination. Further evidence for alternative mRNA splicing was also provided by the results of studies with a catfish clonal B cell line, 3B11, which was observed to express both µ and {delta} messages containing identical VDJ rearrangements (3).

cDNA homologs of {delta} have subsequently been identified in Atlantic salmon, Salmo salar (8), and Atlantic cod, Gadus morhua (9). As in catfish, the {delta} transcripts in these species included Cµ1 as the first C region exon and the {delta} gene was found directly downstream of the µ gene. In addition to the {delta}m and {delta}s cDNAs identified in catfish, evidence for the expression of a catfish {delta}s protein has been obtained; i.e., an anti-peptide Ab specific for the carboxyl terminus of {delta}s identified (by Western analysis) a 180-kDa serum protein, the predicted size of {delta}s (10).

It is now clear that the catfish IgH locus is complex and contains several µ and {delta} genes. The first described and most intensively studied catfish µ gene (3, 11, 12, 13) is termed IGHM1, and the {delta} gene immediately 3' of it is IGHD1. A second µ gene, determined to be a pseudogene and therefore termed IGHM2P, has been described ~725 kb upstream of IGHM1 (14, 15). It was initially believed that secreted and membrane-bound forms of catfish IgM and IgD originated from the IGHM1 and IGHD1 genes. This conclusion was supported by the pseudogene status of IGHM2P, which contains only remnants of exons Cµ1 and Cµ2 (14). Reported here is the evidence that the catfish IgH locus contains three {delta} constant region genes or pseudogenes, each linked to a µ gene or pseudogene. The complete characterization of two of these catfish {delta} genes has led to considerable revision in our understanding of the expression of IgD in this species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Outbred channel catfish (1–2 kg) were obtained from ConAgra (Isola, MS) and second-generation homozygous MHC-matched gynogenetic channel catfish were obtained from the Catfish Genetics Research Unit, U.S. Department of Agriculture-Agricultural Research Service (Stoneville, MS) (16, 17) and maintained in individual tanks (18).

Genomic library construction and screening

Genomic DNA was prepared from erythrocytes of a single outbred catfish and a recombinant catfish genomic library was created in {lambda} phage. Briefly, partially SauIIIA-digested DNA was size-separated by centrifugation on a sucrose density gradient and fragments of ~15–20 kb were ligated and packaged in Lambda EMBL-4 (Stratagene, La Jolla, CA). The library was screened with catfish {delta}2/{delta}3 and {delta}6/{delta}7/{delta} transmembrane (TM)6 probes. Seven hybridizing clones were identified and phage clones, {lambda}IgD2, {lambda}IgD3, and {lambda}IgD6, were chosen for further analyses. Overlapping restriction fragments from each of the three recombinant genomic clones were subcloned into either pUC19 or pBluescript (Stratagene) and completely sequenced on both strands (Biotechnology Resource Laboratory, Medical University of South Carolina, Charleston, SC). Sequences were analyzed using DNAstar (DNAstar, Madison, WI) and Vector NTI Deluxe v4.0.1 (InforMax, North Bethesda, MD).

A recombinant genomic bacterial artificial chromosome (BAC) library was prepared with DNA isolated from brain tissue of a single gynogenetic catfish (S. M.-A. Quiniou, unpublished data). Briefly, high-molecular weight DNA was partially digested with HindIII enzyme and ligated into pBeloBac11 vector (19) (Invitrogen, Carlsbad, CA). Approximately 50,000 bacterial colonies were plated on Luria-Bertani plates containing 12.5 µg/ml chloramphenicol and screened with a catfish {delta}7 probe. Six hybridizing clones, BAC 1–6, were identified; each contained an insert of ~130 kb.

Southern blotting and mapping of BAC clones

Genomic DNA was prepared from erythrocytes of individual gynogenetic or outbred fish by lysis in TES buffer (10 mM Tris (pH 8), 10 mM EDTA, 400 mM NaCl, 0.2% SDS) containing 100 µg/ml proteinase K. After overnight digestion at 37°C the DNA was extracted with 6 M NaCl (20). BAC DNA was isolated using standard alkaline lysis. Genomic DNA (10 µg) or recombinant BAC DNA (0.2 µg) was digested with EcoRI separated on a 1% agarose gel and transferred by capillary action onto Hybond-N+ charged nylon membranes (Amersham Pharmacia Biotech, Arlington Heights, IL) using standard techniques. Hybridizations were performed in UltraHyb hybridization solution (Ambion, Austin, TX) at 42°C as per the manufacturer’s recommended instructions. The membranes were washed four times at high stringency (62°C with 0.2x SSC plus 0.1% SDS) and subjected to autoradiography.

Recombinant BAC clones (clone 2 and 4) were mapped by digesting 1 µg of DNA with PvuI, MluI, ClaI, BssHII, SgrAI, and NotI (New England Biolabs, Beverly, MA) to completion, either separately or in combinations, and separated by pulsed field gel electrophoresis (CHEF-MAPPER; Bio-Rad, Hercules, CA) on 1% agarose gels (Seakem LE; BioWhittaker, Rockland, ME) in 0.5x TBE along with Lambda Ladder PFG marker (New England Biolabs) using the following parameters: current, 6 V/cm; angle, 120 degrees; pulse interval ramping, 5–15 s; run time, 15 h; temperature, 14°C. The sizes of the DNA fragments were calculated using the GelExpert software (NucleoTech, Hayward, CA). The DNA was transferred and hybridized as above.

Probes

Probes for Southern blot and screening were amplified by PCR using AmpliTaq (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s recommended protocol. Typical parameters were as follows: 1 min at 95°C, 1 min at 61°C, and 2 min at 72°C for 30 cycles. The specific µ and {delta} probes (Table IGo) were random prime labeled with [32P]dCTP using the Megaprime labeling kit (Amersham Pharmacia Biotech).


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  		Table I. PCR-derived probes used in library screening and Southern blot analyses

 
Plasmids, transient transfections, and reporter assays

The enhancer-less plasmid pFVH-CAT, containing a goldfish VH promoter and the chloramphenicol acetyl transferase (CAT) reporter gene, and the positive control plasmid pFVH-CAT-ELF11, containing the catfish IgH enhancer Eµ3', have been described by Magor et al. (11). The catfish test construct, E{delta}6.1, was prepared by ligating into pFVH-CAT a 2824-bp HindIII/BamHI fragment that contains the putative enhancer region of the IGHM3P/IGHD3 gene; i.e., nt 5813–8637 of genomic phage {lambda}IgD6. This fragment contains seven octamer motifs and two µE5 motifs that match those in the previously described catfish Eµ3' enhancer (11). Plasmid pCMV-LUC served as an internal control for transfection efficiencies (21). Plasmid DNAs were purified using QiaFilter Plasmid Maxi kit (Qiagen, Valencia, CA).

The catfish clonal B cell line 1G8 was cultured as described (22, 23). For transfection, 3.5 pmol (10–16 µg) of the test plasmids were introduced by electroporation into 1G8 together with 2 µg of pCMV-LUC as an efficiency control. In all samples, pUC19 was used as carrier DNA to adjust the total amount of DNA to 20 µg. Electroporation was as described by Bengtén et al. (21) and experiments were conducted in triplicate. Expression of reporter constructs was measured 30 h posttransfection. Briefly, cells were harvested and washed in PBS and extracts were prepared in 1x cell culture lysis reagent (Promega, Madison WI) for luciferase and CAT assays. Luminescence from pCMV-LUC was used to normalize the activity levels for transfection efficiency with luciferase activity measured in triplicate using the Promega luciferase assay system and a LumiCount (Packard Instrument, Meriden, CT). CAT activity was measured using the liquid phase assay as described by Askovic and Baumann (24). Briefly, cell extracts were incubated with 0.25 µCi [3H]acetyl-coenzyme A (Amersham Pharmacia Biotech) for 2 h. After incubation the 3H-acetylated chloramphenicol was extracted into toluene and quantified by liquid scintillation.

Abs and Western blots

Anti-{delta}s polyclonal Ab (pAb) was produced by immunizing BALB/c mice with a multiantigenic peptide (MAP) (25) comprised of seven copies of the {delta}s carboxyl terminus (FTEETIYFDENKYEQLLTAPSRP). Mice were injected i.p. with 100 µg of peptide in CFA and three subsequent immunizations were given in IFA. Blood was collected from the tail vein. Polyclonal ascitic fluid was generated by injecting immunized mice with 0.5 ml pristane followed 2 wk later with an injection of 2 x 106 nonsecreting myeloma cells SP2/0 Ag-14 (26). Anti-{delta}s mAbs were generated according to standard methods (27). The {delta}S MAP was synthesized by the Biotechnology Resource Laboratory (Medical University of South Carolina).

Serum from individual catfish was separated on 8% SDS-PAGE gels under reducing conditions. The proteins were electrophoretically transferred to nitrocellulose Hybond ECL (Amersham Pharmacia Biotech). The membranes were blocked with 15% fat-free milk in TBS-T (TBS plus 0.05% Tween 20) and incubated with either anti-{delta}s pAb or anti-{delta}s mAb 2E5 (IgG2a{kappa}). Following four washes in TBS-T the membranes were incubated with HRP-conjugated goat anti-mouse IgG and developed using the ECL Western blot detection kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocol.

Purification of serum IgD

The IgD protein was purified from catfish serum using gel filtration and preparative SDS-PAGE. Briefly, 7–9 ml of serum was applied to an XK50/60 column of Sephacryl S-300 (Amersham Pharmacia Biotech) in 0.5 M Tris (pH 8), 0.15 M NaCl, 0.5 mM EDTA, and the IgD-containing fractions were determined by Western blot analyses. These were pooled and concentrated ~5-fold using a Centriprep YM-10 (Amicon, Beverly, MA). Twenty-five microliters (maximum volume of well) of the concentrated IgD pool were then electrophoresed under reducing conditions on a 5% SDS polyacrylamide gel. The gel was stained with Coomassie blue using standard conditions and the {delta} protein band was excised. Gel slices were washed twice in 50% acetonitrile in HPLC-grade water and snap-frozen in liquid nitrogen. Samples were sent to the Harvard Microchemistry Facility (Harvard University, Boston, MA) and were subjected to proteolytic digestion and sequencing. Sequence analysis was performed by microcapillary reversed-phase HPLC nanoelectrospray tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and mapping of {delta} genes in the catfish IgH locus

Previous cloning and sequence analysis of the catfish IgH locus revealed that exons 1 and 2 of an expressed {delta} gene resided ~1.6 kb 3' of the IGHM1 TM2 (µTM2) exon (3). To extend the analysis of this catfish {delta} gene, a recombinant genomic {lambda} phage library from a single outbred fish was screened with probes corresponding to the 5' and 3' regions of catfish {delta} cDNA. Seven recombinant clones hybridized with both probes, and the three with the longest inserts (termed {lambda}IgD2, {lambda}IgD3, and {lambda}IgD6; GenBank accession nos. AF363448, AF363450, and AF363449, respectively) were selected for further analysis. Complete mapping and sequencing of the inserts within these phage revealed that they represented two separate contigs of 11245 and 18486 bp, respectively, which included two distinct, albeit very similar, {delta} genes (Fig. 1Go). Both {delta} genes contained exons for {delta}1, {delta}2, {delta}3, and {delta}4, followed by a tandem duplication of {delta}2-{delta}3-{delta}4, followed by {delta}5, {delta}6, {delta}7 and an exon encoding for either the TM region or the {delta}s.



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  		FIGURE 1. The catfish IgH locus. A, Map showing the six identified C region genes of the catfish. Linkage between two catfish Cµ genes (IGHM1 and IGHM2P) was previously established (15 ). Linkage among IGHM2P, IGHM3P, and the two catfish C{delta} genes (IGHD2 and IGHD3) was established by restriction mapping, pulsed field gel electrophoresis, and Southern blot analyses of recombinant BAC4. Phage clones {lambda}IgD2, {lambda}IgD3, and {lambda}IgD6 were isolated in this study and phage {lambda}12C (3 12 ) and {lambda}C2 (14 ) have been reported previously. B, Physical maps of catfish genomic {lambda} phage clones {lambda}IgD2, {lambda}IgD3, and {lambda}IgD6 showing contigs linking IGHM1 with IGHD1 and IGHM3P with IGHD3. Restriction sites (S, SstI; E, EcoRI) are marked. The overlapping, previously described phage clone {lambda}12C is shown for comparison. Schematics are drawn to scale.

 
The sequence of the {lambda}IgD2 insert overlapped with that of the previously sequenced clone {lambda}12C (GenBank accession no. X79482) (11); therefore, these sequences could be assembled into a contig (Fig. 1GoB) spanning the Cµ (IGHM1) and C{delta} (IGHD1) genes, from the proximal JH region to 2 kb downstream of the IGHD1 TM exon. The second {delta} gene was characterized from a contig of two phage: {lambda}IgD6 and {lambda}IgD3 (Fig. 1GoB). This contig extends upstream of the {delta} gene and includes three remnant exons ({psi}Cµ2, {psi}Cµ3, and {psi}Cµ4) of a pseudo µ gene. Interestingly, the {lambda}IgD6/3 contig did not contain or overlap with the previously described catfish pseudo µ gene (14) now termed IGHM2P. Consequently, there are two µ pseudogenes in the catfish IgH locus, IGHM2P and IGHM3P (described in this paper). To map the organization of the multiple µ and {delta} genes present in the catfish IgH locus, catfish BAC clones hybridizing with probes for {delta}7 were isolated. Southern blots of six EcoRI-restricted BAC clones were sequentially hybridized with different probes for µ, {delta}, and a Tc1 transposable element (Fig. 2GoA). Because {lambda}IgD3, {lambda}IgD6, {lambda}IgD2, and {lambda}12C had been completely sequenced the identity of the hybridizing bands was readily determined. The restriction digests and blot hybridizations demonstrated that one clone (BAC6) contained IGHM1 and IGHD1, i.e., restriction fragments hybridizing with Cµ1, Cµ2, and Cµ3 probes exhibiting sizes corresponding to the expected fragment sizes calculated from the {lambda}12C sequence. Likewise, the sizes of fragments hybridizing with C{delta}1, C{delta}3, and C{delta}7 probes correspond to the expected sizes as determined from the {lambda}IgD2 sequence. The other five clones (BAC1–5) each contained the IGHM3P gene (Fig. 2GoA, open arrows) and BACS 1, 3, 4, and 5 also contained the previously described IGHM2P gene (Fig. 2GoA, open circle). For example, the 5.4-kb band hybridizing with {psi}Cµ3-Cµ4 was distinctive for IGHM3P as predicted by the {lambda}IgD6 sequence, while the 4.8-kb hybridizing Cµ2 band was distinctive for IGHM2P as predicted from the previously described {lambda}2C sequence (14). Thus, the two {psi}µ genes are linked within 130 kb of each other. Furthermore, both {psi}µ genes are linked to {delta} sequences; the linkage of IGHM3P and IGHD3 was established by sequencing of {lambda}IgD6 (Fig. 1GoB) and the linkage of IGHM2P and IGHD2 was suggested by the presence of the "second" Cµ2, C{delta}1, and C{delta}3 hybridizing bands (Fig. 2GoA, open circle) found in BAC1, 3, 4, and 5. In addition, restriction mapping with PvuI, MluI, ClaI, BssHII, SgrAI, and NotI enzymes and Southern blot analyses of BAC4 demonstrated that IGHM2P and IGHD2 are linked on an 11-kb BssH II fragment and IGHD2 and IGHD3 are linked within 70 kb of each other (Fig. 1GoA). Comparison of the recombinant BAC and {lambda} phage hybridization results combined with Southern blot analysis of genomic DNA from 10 individual catfish hybridized with a {delta}1 probe demonstrated that IGHD1 and IGHD3 are present in all catfish studied. Furthermore, they indicate that all Ig C region genes are readily detectable by Southern blot and are included in the map shown in Fig. 1GoA, albeit with some allelic variation in the hybridizing IGHD2 band (Fig. 2GoB). Thus, this evidence clearly indicates that the catfish IgH locus contains three µ and {delta} genes.



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  		FIGURE 2. A, Southern blot analyses of catfish recombinant genomic BAC clones 1–6. BAC clone DNA (0.2 µg) was digested to completion with EcoRI, electrophoresed on 1% agarose gels, and transferred to nylon membranes. The filters were hybridized with 32P-labeled probes for Cµ1, Cµ2, Cµ3, {psi}Cµ3-Cµ4 intron, IpTc1, C{delta}1, C{delta}3, and C{delta}7 (as indicated) and washed at high stringency. Filled arrows, The functional IGHM1 or IGHD1 hybridizing bands; open circle, the IGHM2P or IGHD2 hybridizing bands; open arrows, the IGHM3P or IGHD3 hybridizing bands. The 1.5-kb band hybridizing with C{delta}3 results from the {delta}2-{delta}3-{delta}4 tandem duplication found in IGHD1 and IGHD3. Markers in kilobases are shown to the right. B, Southern blot analysis of catfish genomic DNA. Catfish genomic DNA (10 µg) from one gynogenetic (g58) and nine outbred catfish (1, 3, 5, 7, 32, 55, 77, 82, and 88) was analyzed as above using a catfish C{delta}1 probe. The kilobase marker is indicated to the left and symbols are as in A.

 
Catfish µ and {delta} genes

The complete sequence of {lambda}IgD2, {lambda}IgD3, and {lambda}IgD6 permits direct comparisons of the IGHM1 vs IGHM3P and the IGHD1 vs IGHD3 genes (Fig. 3Go). The three µ genes are very different, i.e., the IGHM1 gene is clearly functional (12), whereas IGHM3P and IGHM2P (14) are highly degenerate pseudogenes. IGHM3P contains three remnant µ exons, {psi}Cµ2, {psi}Cµ3, and {psi}Cµ4, with a remnant of a Tc1/mariner transposable element located between {psi}Cµ2 and {psi}Cµ3. The {psi}Cµ2 exon of IGHM3P is very similar but not identical to the {psi}Cµ2 of IGHM2P (Fig. 4Go). Both contain five stop codons and, except for the first conserved 25 amino acids, are out of frame as compared with the functional Cµ2 of IGHM1. In contrast, the introns preceding the Cµ2 exons are highly conserved. The location of the Tc1 element of IGHM3P is identical to the location of the Tc1 element termed IpTc2 described in IGHM2P (14). However, the Tc1 element of IGHM3P is more complete because it possesses coding blocks A through F and is flanked by terminal repeats (29). IpTc2 lacks terminal repeats and contains only coding block E and part of block D. The {psi}Cµ3 and {psi}Cµ4 remnant exons of IGHM3P are located directly 3' of the Tc1 element and Southern blot analysis suggests that they are also present in IGHM2P. The {psi}Cµ3 remnant is in frame and 55% identical at the nucleotide level to functional Cµ3, with conservation of encoded amino acid residues throughout the domain. However, an 18-aa stretch in the second half of the Cµ3 domain, including the Cys necessary for the interchain disulfide bond, is missing. The {psi}Cµ4 exon consists of only 108 nt corresponding to the first third of the functional Cµ4 (79% identical at the nucleotide level) but does not encode any Cys residues (Fig. 5Go). No identifiable µTM exons are found 3' of the Cµ4 exon remnant, but a region showing strong sequence conservation with the Eµ3' transcriptional enhancer of IGHM1 (11) is present (Figs. 3Go and 6GoA). This IGHM3P-associated enhancer region contains two octamers and a µE5 motif (O10, O11, and E5-2) that are known to comprise the minimal functional unit of Eµ3' critical for enhancer function (30). In addition, this putative enhancer contains five other motifs conserved in the Eµ3' enhancer: four octamers and one Eµ5 motif, plus two additional unique octamer motifs. Furthermore, this putative enhancer was tested for activity by transient transfection. To this end a BamHI/HindIII fragment encompassing nt 5813–8637 of {lambda}IgD6 was cloned into the pFV-CAT reporter plasmid (11). This catfish IGHM3P/IGHD3 enhancer construct was transfected by electroporation into the catfish clonal autonomous B cell line 1G8 together with the control plasmid pCMV-LUC and assessed for enhancer activity. This fragment exhibited strong enhancer activity, which is greater than that of the catfish Eµ3' (pFV-CAT-ELF11; 11) from the IGHM1 gene (Fig. 6GoB).



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  		FIGURE 3. Comparisons of two µ{delta} gene regions of the catfish IgH locus. Inverted repeats of the IpTc1/Mariner elements are marked by arrows and E marks the IgH enhancer regions. Black indicates sequence similarity (>79% identity) between the two regions, gray indicates that the sequences are different (<55% identity), and white represents areas not present in one gene but present in the other. Short interspersed repetitive elements (SINE) are marked. The IGHM1/IGHD1 map is based on the previously described {lambda}12C (12 ) and {lambda}IgD2. Schematics are to scale.

 


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  		FIGURE 4. Nucleotide and amino acid sequence alignments of the catfish IgH genes and Tc1 elements. A, Comparison of the {psi}Cµ2 and functional Cµ2 exons. The IGHM3P sequence initiates 5' of {psi}Cµ2 and nt 1459–3218 of {lambda}IgD6 are compared with nt 3881–4870 of {lambda}12C (IGHM1) (12 ) and nt 8996–9878 of {lambda}2C (IGHM2P) (14 ). Exon boundaries are marked by {downarrow} and the start of the exons are labeled C2. Periods indicate identity and dashes show gaps. Stop codons are indicated by asterisks. The inferred amino acid sequences of Cµ2 and the {psi}Cµ2 exons are shown below the second base of the codon; those that are identical to the functional Cµ2 are in bold. The nucleotide numbers and {lambda} phage clone names are to the right.

 


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  		FIGURE 5. Alignment of the nucleotide sequence of catfish IGHM3P and IGHM1 genes comparing the {psi}Cu3, {psi}Cu4, and functional Cµ3 and Cµ4 exons. Nucleotides 4719–5389 of {lambda}IgD6 and nt 4871–5883 of {lambda}12C are shown. Exon boundaries are marked by {downarrow} and the start of the exons is labeled C3 or C4. Periods indicate identity and dashes show gaps. The inferred amino acid sequences of the {psi}µ exons and the Cµ3 and Cµ4 exons are shown below the second base of the codon; those that are identical are in bold. The nucleotide numbers and {lambda} phage clone names are to the right.

 


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  		FIGURE 6. A, Map of the IgH chain enhancer regions of IGHM3P/IGHD3 and IGHM1/IGHD1. Black indicates sequence similarity (>79% identity) between the two regions, gray indicates sequence differences (<55% identity), and white represents areas not present in one gene but present in the other. O, Octamer; O*, octamers only present in IGHM3P/IGHD3; E3, µE3 motif; E5, µE5 motif. Octamers are numbered by their order of occurrence in {lambda}12C (according to Ref. 11 ). B, Relative CAT activity resulting from transient transfection of catfish IgH enhancer regions into catfish 1G8 B cells. Constructs contained the previously described catfish Eµ3' enhancer (pFV-CAT-ELF11) (11 ) or the catfish IGHM3P/IGHD3 enhancer region (pFV-CAT-E{delta}6.1). Results are the average of two transfections conducted in triplicate. CAT activities are normalized in correlation to the activity of the cotransfected CMV-LUC plasmid and given as mean ± SD, relative to the transcriptional activity of the empty vector control (pFVH-CAT), which is set to 1.

 
In marked contrast to the major sequence differences found within the three µ genes, the two {delta} genes compared here are highly conserved. They exhibit a high degree of identity at both the exon and intron levels (Fig. 3Go and Table IIGo) and both appear to be functional, because all exons have consensus splice donor and acceptor signals and are in frame. The duplicated exons within each gene also exhibit strong homologies with nucleotide sequence identities ranging from 91 to 100% (Table IIGo). The only exception involves comparing {delta}4.1 to {delta}4.2 in either locus: {delta}4.1 is 5 aa shorter than {delta}4.2 and they are 76 and 62% identical at the nucleotide and amino acid levels, respectively. The splice acceptor/donor sequences of the exons in IGHD3 are identical to those found in the IGHD1 gene, except for the acceptor splice site of {delta}6. A different {delta}6 acceptor splice site in IGHD3 appears to be formed as a result of 7 nt differences (underlined), TTTGTATTTATTCAG/AC as compared with TATTTGTAG/GCTCAGAC. The shift in splice site usage (confirmed by cDNA sequencing) adds two additional amino acids to the exon. This IGHD3 {delta}6 splice site reflects the introduction of an AG dinucleotide and the preferential use of the more 5' AG as a splice acceptor site (31, 32). In toto there are 24 insertions/deletions between these two {delta} genes, with most being short, i.e., 14 are <5 bp. The largest insertion was found in the IGHD1 gene where the {delta}6/{delta}7 intron contains an 809-bp insertion flanked by TATA repeats (Fig. 3Go).


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  		Table II. Comparisons of the nucleotide and amino acid sequences of catfish {delta} exons and the nucleotide sequences and lengths of {delta} introns

 
The most significant differences between the catfish IGHD1 and IGHD3 genes reside in their terminal exons (Fig. 7GoA). The IGHD1 gene ends with an exon encoding a typical Ig-like TM region, whereas the IGHD3 gene terminates with the shorter {delta}s exon. Thus, the truncated {delta}s cDNA sequences previously obtained were not derived by alternative RNA processing from a primary transcript of the IGHD1 gene (3). Interestingly, the nucleotide sequences encoding the first 15 aa of the {delta}s and {delta}TM exons are almost identical and, when {delta}s and {delta}TM are aligned, the nucleotides encoding the last 9 aa of {delta}s match with the 3' untranslated region of {delta}TM, albeit in the second reading frame (Fig. 7GoA). Thus, {delta}s differs from {delta}TM primarily by a 121-bp deletion. The presence of only a single {delta}TM exon was unexpected because the TM regions of most other Igs, except IgA, are encoded by two exons (reviewed in Refs. 1 and 2). A potential 2-aa {delta}TM2 (VK) exon is found 857 nt downstream of IGHD1 (Fig. 7GoB) and a potential splice donor site is located in the {delta}TM exon (marked in Fig. 7GoA). However, RT-PCR protocols performed using forward primers for Cµ1 and various {delta} exons ({delta}1, {delta}2, {delta}3, {delta}5, {delta}6, and {delta}7) in combination with reverse primers for regions 3' of {delta}TM2 yielded no products; therefore, it was concluded that {delta}TM2 is a pseudoexon, designated {psi}{delta}TM2.



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  		FIGURE 7. A, Schematic and alignment of the nucleotide and inferred amino acid sequences of catfish {delta}s and {delta}TM. Black indicates sequence similarity; hatching indicates the sequence that encodes only for {delta}s. The {delta}TM sequence, which is absent in {delta}s, is shown in gray. The splice between {delta}7 and the terminal exons is marked by "/." Identities are indicated by periods, gaps by dashes, and stop codons by asterisks. Inferred amino acids are below the second base of each codon. The potential splice site in {delta}TM is double-underlined and polyadenylation sites are underlined. B, Sequence of the {psi}{delta}TM2 exon found in IGHD1. The potential splice acceptor site is marked by "/." Nucleotide numbers are shown to the right.

 
To determine whether the IGHD3 gene is expressed, polyclonal mouse antisera (pAb anti-{delta}s) and a mAb (2E5) were generated against a MAP of the catfish {delta}s carboxyl terminus. Initial Western blot analyses revealed that both Abs reacted with a serum protein of ~180 kDa. Moreover, this reactivity could be completely inhibited by the addition of excess synthetic peptide (10). Identification of the 180-kDa protein as {delta} was confirmed by protein purification and peptide sequencing. Fig. 8GoA shows a SDS-PAGE and a corresponding Western blot of fractions obtained by gel filtration of catfish serum on Sephacryl S-300. Aliquots of concentrated fraction 28 were electrophoresed on 5% SDS-PAGE (Fig. 8GoB), and the presumed {delta} protein band was excised from each lane and subsequently sequenced by microcapillary reversed-phase HPLC nanoelectrospray tandem mass spectrometry. Table IIIGo lists the {delta} peptide sequences obtained by mass spectrometric analysis. Three of the peptides, one each from {delta}1, {delta}5, and {delta}6, could only have originated from IGHD3; i.e., each contains specific amino acid residues encoded by the exons of that gene. Amino acids that are specific and conserved for the {delta}2.1, {delta}2.2, {delta}3.2, and {delta}4.2 exons found in both {delta} genes were also identified (Table IIIGo), suggestingthat the duplicated {delta} exons are expressed. Based on densitometry of Coomassie blue-stained gels the concentration of {delta} protein in catfish serum is ~0.04 mg/ml.



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  		FIGURE 8. Catfish serum contains IgD protein. A, Samples from unseparated serum (S) and fractions 27–31, obtained by gel filtration of catfish serum on Sephacryl S-300, were separated under reducing conditions by 8% SDS-PAGE and stained with Coomassie blue (top). Western blot analysis was performed with a duplicate gel using the anti-{delta}s mAb 2E5 (bottom). B, Aliquots from fraction 28 were separated under reducing conditions by 5% SDS-PAGE. The {delta}s-containing band (as indicated by the arrow) was excised and sequenced by tandem mass spectrometry.

 

View this table:
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  		Table III. Tandem mass spectrometry peptide sequences corresponding to the inferred amino acid sequences of catfish {delta} exons

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies reported in this paper, in conjunction with previous reports (3, 12, 14, 15), reveal a previously unexpected complexity in the catfish IgH locus; i.e., three µ genes and three {delta} genes are present. These almost certainly arose as the result of large gene duplication events. The 3'-most µ and {delta} genes in the locus (IGHM1 and IGHMD1) are fully functional and encode H chains of catfish IgM and IgD (3, 11, 12, 13). In the 5' region of the locus (~725 kb upstream of IGHM1), the previously reported {psi}µ gene (IGHM2P), associated with a germline-fused VDJ and additional VH and JH elements (14, 15), was found to be linked to a {delta} gene (IGHD2) which has not yet been sequenced. Downstream of IGHD2 a third IgH chain gene cluster was identified, which contains a second {psi}µ gene (IGHM3P), a strong enhancer, and a functional {delta} gene (IGHD3). The presence of transposable elements, particularly Tc1/mariner-like elements (which are highly represented in catfish) (33), within these IgH genes is quite likely related to the duplication events that produced the complex structure of the catfish IgH locus. It has been suggested that remnant Tc1 elements, mobilized by transposases encoded elsewhere in the genome, played a role in the evolution of the vertebrate IgH locus (34) and may also have been involved in the large scale duplications of the catfish IgH locus. It is also likely that Tc1 elements were responsible for the disruptions of structure that have occurred in the two pseudo µ genes. For example, the deletion of µTM1, µTM2, and part of the Eµ3' enhancer region in IGHM3P is presumably the result of excision of the IpTc1 originally present in this region, followed by DNA repair.

In stark contrast to the great disparity between the catfish duplicated µ genes, the two duplicated {delta} genes (IGHD1 and IGHD3) are remarkably conserved. The major difference between the two genes involves the terminal exons: the IGHD1 gene encodes a polypeptide with a typical Ig cytoplasmic tail, whereas the terminal exon of the IGHD3 gene encodes a terminal secretory tail. The finding of cDNAs containing these two alternative terminal exons clearly indicates that both genes are transcribed (3). In a previous report it was suggested that catfish IgD, as now considered to be encoded by the IGHD1 gene, may function as a B cell Ag receptor. Its sequence relatedness, location immediately downstream of the functional µ gene, and coexpression by alternative mRNA processing pathways with µ in some B cells are consistent with catfish {delta} being a true homolog of mammalian {delta} (3). The only evidence to date for a functional IgD TM protein is the existence of full-length {delta}m transcripts encoded by the IGHD1 gene. All catfish full-length {delta} cDNAs identified to date consist of a rearranged VDJ spliced to Cµ1, the {delta}1–{delta}7 domains, and {delta}TM. The catfish {delta}TM encodes for a typical Ig-like TM region and there is no indication from the sequence data that it could not be expressed or function as an Ag receptor on the B cell surface. The finding of a second functional {delta} gene encoding the previously reported putative secreted form of {delta} (3) was unexpected. Coomassie blue staining and Western blot analyses using pAb and mAb specific for the C-terminal {delta}s revealed that {delta} polypeptides are found in normal catfish serum at levels similar to IgD in humans (Fig. 8Go and Refs. 10 and 23). The origin of serum IgD from the IGHD3 gene was subsequently confirmed by protein purification and tandem mass spectrometry peptide sequencing, although the N-terminal sequence of serum IgD was not determined. Catfish full-length {delta}s transcripts have also not yet been identified. 5'-RACE approaches have identified the longest {delta}s clones as beginning with 110 bp of an as-yet-unidentified exon spliced to {delta}1 (E. Bengtén, unpublished observations). Because IGHD3 is located 5' of the IGHM1 and IGHD1 genes, {delta}s may not use the same VDJ rearrangements used by IGHM1 and IGHD1 to form µm, µs, and {delta}m. Therefore, if {delta}s contains a VDJ rearrangement it could use the VH segments 5' of the IGHM2P gene (14). If this is the case then {delta}m and {delta}s may not be produced by the same cell. However, it cannot be ruled out that {delta}m and {delta}s share the same VDJ rearrangements by a mechanism of intrachromosomal homologous recombination as demonstrated for Ig genes in mouse hybridomas (35, 36). It is also possible that separate short sterile transcripts of VDJ and CH genes could be joined by a transplicing mechanism as documented for Ig isotypes in transgenic mouse models (37, 38). At this time it is not known which of these possibilities are operative in channel catfish. Similarly, it is not known whether the IGHD2 gene, identified only by Southern blot hybridization (Fig. 2Go), encodes a functional polypeptide.

From the results of previous studies and those presented here, it can be concluded that the downstream C genes of the catfish IgH locus (IGHM1 and IGHD1) are most likely fully functional, encoding µm, µs, and {delta}m. The upstream genes of the locus (IGHM2P, IGHM3P, IGHD2, IGHD3) most probably resulted from a large duplication event that included most (if not all) of the original locus and that was followed by a second duplication of a smaller region. The upstream genes derived from hypothetical ancient duplication events include multiple Ig elements that are apparently expressible: these include VH genes, a JH element, a fused VHDJH, and, as shown here, an intact {delta}s gene and a functional enhancer. Whether or not the IGHD3-encoded {delta} polypeptides function in the immune response is currently unresolved.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (R01AI-19530 and R01 GM62317), the National Science Foundation (MCB 9807531), and the U.S. Department of Agriculture (99-35204-7844). Back

2 Address correspondence and reprint requests to Dr. Eva Bengtén, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. E-mail address: ebengten{at}microbio.umsmed.edu Back

3 Current address: Catfish Genetics Research Unit, U.S. Department of Agriculture-Agricultural Research Service, Stoneville, MS 38776. Back

4 Current address: Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305-5156. Back

5 Current address: Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824. Back

6 Abbreviations used in this paper: TM, transmembrane; BAC, bacterial artificial chromosome; MAP, multiantigenic peptide; CAT, chloramphenicol acetyl transferase; pAb, polyclonal Ab. Back

Received for publication February 19, 2002. Accepted for publication June 26, 2002.


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 Results
 Discussion
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