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The Journal of Immunology, 2003, 170: 2573-2581.
Copyright © 2003 by The American Association of Immunologists

The T Cell Receptor {beta} Locus of the Channel Catfish, Ictalurus punctatus, Reveals Unique Features1

He Zhou2,*,{dagger}, Eva Bengtén*, Norman W. Miller*, L. William Clem* and Melanie Wilson3,*

* Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216; and {dagger} The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, a series of clonal alloantigen-dependent T cell lines established from the channel catfish revealed distinctly different TCR{beta} rearrangements. Here, a follow-up study of the junctional diversity of these TCR gene rearrangements focuses on characterization of the genomic organization of the TCRB locus. Surprisingly, a total of 29 JB genes and two substantially different CB genes were identified downstream of a single DB gene. This is in contrast to the situation in mammals, where two clusters of a DB gene, six or seven JB genes, and a CB gene are found in tandem. The catfish CB genes are ~36% identical at the amino acid level. All 29 catfish JB gene segments appear functional. Thirteen were used in the 19 cDNAs analyzed, of these eight were used by the 11 catfish clonal alloantigen-dependent T cell lines. As might be expected, CDR3 diversity is enhanced by N-nucleotide additions as well as nucleotide deletions at the V-D and D-J junctions. Taken together, compared with that in mammals, genomic sequencing of the catfish TCR DB-JB-CB region reveals a unique locus containing a greater number of JB genes and two distinct CB genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Full-length cDNAs encoding TCR {alpha}- and {beta}-chains were previously identified and characterized in the channel catfish, Ictalurus punctatus (1). The catfish V{alpha} and V{beta} sequences each shared hallmark residues with the V{alpha} and V{beta} genes of other vertebrates, and as in other vertebrates, the catfish C{alpha} and C{beta} region sequences exhibited distinct Ig, connecting peptide (CP),4 transmembrane (TM), and cytoplasmic (CYT) domains. In addition, Southern blot and sequence analyses indicated that each of the catfish loci is arranged in a translocon organization with multiple V elements and a single constant (C) region for TCR{alpha} and possibly two C regions for TCR{beta}. Two different TCR C{beta} cDNA sequences with 97% nucleotide identities were observed, but whether they represented alleles or tandemly arranged genes was not resolved.

More recently, cell lines cloned from alloantigen-stimulated PBL have permitted the demonstration of bona fide CTL in catfish (2). Fourteen alloantigen-dependent T cell lines were developed by limiting dilution cloning of catfish PBL isolated from a fish (designated #32) immunized with the catfish autonomous long term B cell line 3B11 (3). These T cell lines are considered nonautonomous, since they require periodic restimulation with the alloantigen (irradiated 3B11cells), and each T cell clone expresses single unique V{alpha} and V{beta} rearrangements, indicating the cell lines are derived from different precursors. Thirteen of the clones are CTL, with 10 being alloantigen-specific cytotoxic T cells, i.e., they specifically proliferate in response to and kill 3B11s. Three other clones are alloantigen-broadly-specific T cells, i.e., they proliferate in response to and kill some, but not all, allogeneic cells in addition to 3B11 cells. The final T cell clone contains noncytotoxic T cells that specifically proliferate in response to 3B11 stimulation; it is speculated that these are alloantigen-specific Th cells. The results of "cold" target inhibition studies suggest that both types of the catfish CTL clones recognize their targets via a single receptor (4). These 13 cell lines are the first CTL lines described in any ectotherm, and the ability to clone them provides a unique opportunity for understanding cell-mediated responses in teleosts (5).

In the study presented here a catfish {lambda} genomic library generated from erythrocytes of fish #32 was screened for TCRB genes to 1) determine the genomic organization of the TCRB locus and 2) examine the CDR3 variability of catfish TCR{beta} cDNAs. The use of this genomic library is a good model for examining DB/JB usage in catfish because fish #32 was the one used to generate the aforementioned 14 alloantigen-dependent T cell clones. Since the TCR{alpha} and {beta} V gene rearrangements of these clones are known, direct analyses of DB-JB usage and junctional diversity in catfish TCR{beta} CDR3 regions are facilitated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Genomic library construction and screening

Genomic DNA was prepared from catfish #32 erythrocytes by lysis in TES buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 400 mM NaCl, 0.2% SDS) containing 100 µg/ml of proteinase K according to Miller et al. (6). After overnight digestion at 37°C, the DNA was extracted with 6 M NaCl, precipitated and dissolved in 1 X TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). The genomic library was prepared in {lambda}DASH II/BamHI vector (Stratagene, La Jolla, CA). 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 into {lambda}DASH. The unamplified library contained ~1.1 x 106 recombinants, and it was screened using standard techniques (7) with a catfish TCR C{beta}-region probe random prime labeled with 32P-dCTP (Megaprime Labeling Kit; Amersham Pharmacia Biotech, Arlington Heights, IL). This probe was amplified from catfish TCRB cDNA using AmpliTaq (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s recommended protocol. The parameters were 1 min at 95°C, 1 min at 65°C, and 2 min at 72°C for 30 cycles. The specific primers used are listed in Table I.


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  		Table I. PCR primers used in Southern blot analyses

 
Three positive phage clones, {lambda}24b, {lambda}16b, and {lambda}8b, were chosen for further analyses. Overlapping restriction fragments from each of the recombinant phage were subcloned into pUC18 EcoRI Ready to Go (Amersham Pharmacia Biotech) or into pUC18 cut with PstI or HindIII. Plasmids were sequenced on both strands using universal forward and reverse and gene-specific primers. Sequencing was performed either in-house, at Jackson, or at the Biotechnology Resource Laboratory at the Medical University of South Carolina. Sequences were analyzed using DNAstar (DNAstar, Madison, WI) and Vector NTI Delux v4.0.1 (InforMax, North Bethesda, MD).

RT-PCR and Southern blotting

TS32 and 3B11 cell lines were assayed for TCR and Ig message expression, and catfish PBL, liver, spleen, pronephros (head kidney), mesonephros (trunk kidney) and gut/intestine tissues were assayed for TCRB1 and TCRB2 expression using RT-PCR. Briefly, total RNA was prepared from cell lines and tissues from a single outbred catfish using RNAzol B (Tel-Test, Inc., Friendswood, TX) or a QuickPrep Total RNA Extraction Kit (Amersham Pharmacia Biotech) and were treated with DNase (Invitrogen Technologies, Carlsbad, CA) at 1 U/µg of RNA according to the manufacturer’s recommended protocols. Approximately, 1 µg of total RNA was converted into first-strand cDNA using 50 ng of an oligo(dT) primer with 200 U of reverse transcriptase (SuperScript II; Invitrogen, San Diego, CA) according to the manufacturer’s recommended protocol. One percent of the first-strand reaction was used as template in RT-PCR. The reaction mixture (50 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 150 mM dNTP, and 0.5 µg of specific forward and reverse primers. Two units of AmpliTaq (PE Applied Biosystems) was added, and 30 cycles of amplification were performed (94°C for 30 s, 65°C for 30 s, 72°C for 1 min). The parameters for amplifying actin and TCRB2 were as described above, except 53°C was used as the annealing temperature for both. The specific primers used are listed in Table I. All PCR products were verified by cloning and sequencing.

Genomic DNA was isolated from outbred catfish #1 and #32, the autonomous long-term T cell line G14D (10), the long term B cell lines 1G8 (11) and 3B11 (3), and the nonautonomous T cell lines TS32.5, TS32.15, and TS32.17 (2). The DNA was prepared as described above by lysis in TES buffer. Ten micrograms of genomic DNA was digested with EcoRI, separated on 1% agarose gels, and transferred by capillary action onto Hybond-N+-charged nylon membranes (Amersham Pharmacia Biotech) using standard techniques. Hybridizations were performed at 65°C with a specific catfish TCR CB1 and MHC class II {beta}2 probe (see Table I). Membranes were washed at high stringency (65°C with 0.2x SSC and 0.1% SDS) and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Catfish TCR{beta} locus

Previous cloning and sequencing of catfish TCR{beta} cDNAs identified two different catfish CB region sequences that were 97% identical at the nucleotide level. To determine whether these two sequences represented two TCRB loci or alleles, a recombinant genomic {lambda} phage library from an outbred catfish (designated #32) was screened with a catfish TCR CB region probe. Three phage clones were selected for analysis; complete mapping and sequencing revealed that two ({lambda}24b and {lambda}16b) overlapped, forming a contig of 19,570 bp (Fig. 1A). This contig included a single DB gene, 29 JB genes, and a CB gene corresponding to the C{beta}2 cDNA sequence originally described (1). The third phage ({lambda}8b), which has been only partially sequenced, contained the 29 JB genes and the previously described C{beta}1 C region. Fig. 1B shows an alignment of the C region exons found in {lambda} phage 16b and 8b. Both C region genes consist of four exons spaced by short introns with conserved splice signal sequences (12) at each intron-exon boundary. The first exon encodes a 112 (C{beta}1) or a 113 (C{beta}2) amino acid Ig domain, the second exon encodes an eight amino acid CP, the third exon encodes the TM, and the fourth encodes for the positively charged short CYT. There are only 17 nucleotide differences between these C regions, i.e., 15 in the Ig domain and two in the CP/TM intron. {lambda}8b and the contig formed by {lambda}24 and {lambda}16b are almost identical; the only sequence differences found in an ~7.5-kb span consist of one nucleotide in the JB13 segment, 23 intron nucleotide changes, differences in the lengths of two microsatellite repeats, and a 30-bp intronic insertion found only in phage {lambda}8b. These high sequence identities among the three phage combined with Southern blot analyses of four outbred and one gynogenetic catfish using JB and CB probes (data not shown) indicate that the two previously described catfish C{beta}1 and C{beta}2 cDNA sequences (1) represent alleles. Therefore, it is concluded that they are encoded by the TCRCB1 gene, and the alleles are designated TCRCB1*1 and TCRCB1*2.



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  		FIGURE 1. The catfish TCR DB-JB-CB region (A) Schematic representation of catfish TCR{beta} genomic phage clones. A 19.8-kb sequence was assembled from two overlapping phage clones: {lambda}CB24b and {lambda}CB16b. The boxes show the locations of CB1 and CB2. Vertical bars indicate D{beta}- and J{beta}-coding sequences. JB segments were named numerically (JB1–29) according to their location in the germline. E marks EcoRI sites. The lower dashed line represents phage clone {lambda}CB8b (17 kb), which contains the allele TCRCB1*1. The schematic is drawn to scale. The GenBank accession number for contig {lambda}24b and {lambda}16b is AF410785. B, Alignment of the genomic sequences of the two catfish TCRC1B alleles. Coding sequences are in bold, deduced amino acid sequences are shown below the nucleotide sequences, and exon names are shown above nucleotide sequences. Splice sites are overlined, and the poly(A) signal, AATAAA, is underlined. Dots indicate identity, and dashes are gaps introduced to achieve maximal alignment. C, Deduced amino acid alignment of the two catfish TCR CB genes. Exon names are shown above the amino acid sequence; dots indicate identity, and dashes show gaps.

 
Interestingly, a second CB region is found ~4 kb 3' of TCRCB1. This second CB gene is also comprised of an Ig domain and CP, TM, and CYT regions, and it is designated TCRCB2 (accession no. AF410785). However, this gene is not as compact as TCRCB1. For example, in TCRCB2 the Ig domain/CP intron is 776 bp in length compared with 112 bp in TCRCB1. TCRCB2 has ~36% identity at the amino acid level with TCRCB1 (Fig. 1C), and as in catfish TCRCB1 and other teleost TCRB genes (1, 13, 14), there is no cysteine in the CP region, which presumably would be involved in interchain disulfide bonding. At the present time the exact exon/intron boundaries can only be inferred from RT-PCR of different catfish tissues using the TCRCB2 Ig domain forward and the presumed 3' untranslated region (3'UTR) reverse specific or TM reverse primer (Fig. 2). In all catfish tissues examined the TCRCB2 message is expressed at a lower level than the TCRCB1 message, and the message level for TCRCB2 appears to be highest in intestine. Additional faint bands above and below the major TCRCB2 are also seen in the intestine RT-PCR. Curiously, both contain a TCRCB2 Ig domain that is missing 127 bp (nt 17,313–17,440). The upper band is unspliced, consisting of truncated Ig domain/CP/intron/TM; the lower band is correctly spliced. Whether these products represent an as yet unidentified TCRCB2 pseudogene or are a PCR artifact has not been determined. Full-length cDNA transcripts for TCRCB2 have not yet been isolated.



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  		FIGURE 2. RT-PCR analysis of RNA encoding TCRCB1 and TCRCB2 in different catfish lymphoid tissues. Total RNA was isolated from catfish PBL, pronephros (Pro), mesonephros (Meso), intestine (Int), spleen (Sp), and liver and subjected to RT-PCR using TCRCB1 and TCRCB2 primers (upper panel). The amplified products (see Materials and Methods for primers used) were electrophoresed on a 1.0% agarose gel and stained with ethidium bromide. Catfish actin was amplified as a control successfully in all tissues except mesonephros (lower panel). HaeIII {Phi}x174 DNA markers are shown at the left.

 
The catfish DB and 29 JB segments are located 5' of TCRCB1. The JB segments are named numerically according to their location in the germline, i.e., the segment immediately downstream (595 bp) of DB is JB1, and JB29 is directly 5' (2160 bp) of CB1. The 29 JB genes are found within a region spanning ~8.4 kb. Each of the JB segments is similar in size, ranging from 48–57 nucleotides in length, and all have identifiable recombination signal sequences (RSS) (15). They can be placed into eight groups based on their sequence similarity and RSS identity. The groups are assigned according to size. Groups I and II contain the largest number of JB genes, with eight members each (Fig. 3, A and B). Group III contains five JB genes, and groups IV and V contain three and two JB genes, respectively. The remaining three JB gene segments (JB1, JB7, and JB28) are placed in the single-member groups VI, VII, and VIII. The JB genes of the different groups are interspersed, except for members of group III, in which JB2 through JB6 are found consecutively in the germline (Fig. 4A). Phylogenetic trees constructed using neighboring-joining (16) and unweighted pair group with arithmetic mean (17) methods support this eight-group classification (data not shown). Within each group the more 3' region of the JB segment is the most conserved; this sequence encodes for framework 4 of the V region. All JB segments contain the conserved framework 4 motif F-G-X-G that is found in most TCR and Ig light chain genes of other animals and is considered structurally important for stabilizing V region conformation, in that it forms the glycine bulge (18). The heptamers, nonamers, and 12-bp spacers within a group clearly show strong identity, suggesting that the segments are the result of gene duplication events (Fig. 4A). The catfish DB gene is located 558 bp 5' of JB1. It is identical in sequence to the single DB gene found in rainbow trout (19, 20) and to human, mouse, rat, and Xenopus DB1 (21, 22, 23, 24). Like these DB genes, it is flanked by highly conserved heptamer and nonamer sequences separated by a 12-bp (5') and a 23-bp (3') spacer (Fig. 3C). The high sequence identities between these DB genes of the different species also extend to the 12-bp (42–75%) and 23-bp (50–83%) spacers. As in the other vertebrates, the catfish DB gene can be used in all three reading frames and encodes one to three glycines depending upon the reading frame. Glycine residues are a typical feature of TCR{beta}, TCR{delta}, and IgH chain V region sequences; they shape the structure/conformation of the Ag-binding CDR3 loop by forming {beta} turns (25).



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  		FIGURE 3. Nucleotide and amino acid sequences of catfish JB and DB genes. A, RSS and nucleotide sequences of catfish JB genes. JB segments are grouped according to RSS and JB sequence identities. Dots indicate identity. B, Deduced amino acid sequences of catfish JB genes. Amino acids are in single letter code; dots indicate identity, and dashes are gaps introduced to achieve maximal alignment. C, Catfish DB gene flanked by RSS. The DB coding sequence is in bold, and deduced amino acids in all three reading frames are shown in single letter code. The RSS is marked.

 


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  		FIGURE 4. Organization of catfish JB genes. A, Schematic representation of catfish JB groups in the germline. The JB genes are shaded according to their group. B, Possible duplication scheme of the catfish JB gene segments. Labeled arrows represent group III tandem duplication (a), group VI-group I block duplications (b), group I-group II JB gene block duplications (c), and group V JB 24 and JB 29 duplication (d). JB genes segments are numbered, and X represents the putative deleted JB segment.

 
The 29 JB intron sequences (named according to the JB exon found directly 3') were aligned using the Clustal method (26). Overall, the introns exhibited low sequence identity when compared with each other. However, exceptions were found within the group I and II introns. The group I introns (introns 12, 14, 16, and 19) exhibited ~70% identity with each other, and the group II introns (introns 11, 13, 15, and 17) had sequence identities with each other ranging from 59–74%. Of the 29 JB introns, only one (intron 18) could be considered unusual in that it appeared chimeric. It had low overall sequence identity (35–44%) with other group II introns and high identity (66–79%) with group I introns. This high identity was due to the first 164 bp, the remaining 97 bp aligned with the group II introns 11, 13, 15, and 17 with identities ranging from 66–75%.

The high sequence identities within a particular JB group and the interspersed pattern of the JB gene segments suggest that this region of the TCRB locus has undergone a series of gene duplications (Fig. 4B). For example, the introns of the group I JB genes JB9, JB23, and JB26 are more similar to each other than to the introns of the other group I JB genes, i.e., 49–57% compared with 32–44%. They are each linked to a group IV JB gene, JB8, JB22, and JB25, respectively, indicating possible block duplications. Another example of presumed block duplication involves JB10-JB19 genes from groups I and II. These JB segments most likely arose from a series of duplications consisting of a single ancestral group I JB gene-intron plus a single ancestral group II JB gene-intron. For example, JB10-intron 11 and JB11-intron 12 duplicated to form JB12-intron 13 and JB13-intron 14 and so on. (Fig. 4B). During this process it is quite possible that a group I JB gene was deleted, giving rise to the chimeric intron 18 and the tandem group II JB segments JB17 and JB18. It is also possible that the five group III JB genes, JB2–JB6, resulted from a separate duplication event, since they are in tandem and have 75–80% sequence identity with each other. However, their introns show low identity (34–53%), perhaps indicating that this duplication occurred earlier than the putative group I/group II duplications.

Catfish TS32 T cell lines

The catfish TS32 alloantigen-dependent CTL clones were first characterized by 1) expression of TCRA, TCRB1, and Igµ genes using RT-PCR; and 2) for allospecific cytotoxic activity in 51Cr release assays (2). A representative RT-PCR analysis for one of the CTL clones, TS32.15, is shown in Fig. 5. This cell line expresses message for both catfish TCR{alpha}- and {beta}-chains, but not for catfish Igµ-chains. In contrast, the catfish long term B cell line 3B11, which is the cell line used as the immunogen for catfish #32, expresses message for catfish Igµ, but not for TCR. Southern blot analyses of the catfish CTL clones were performed to examine TCRB1 gene rearrangements. Fig. 6 is an example comparing representative CTL clones TS32.5, TS32.15, and TS32.17, which have different TCRB1 patterns indicative of different rearrangements. There are two different hybridizing bands in each CTL compared with the two similarly sized larger hybridizing bands of catfish #32 germline DNA. Similarly, the two hybridizing bands of G14D T cells are different from the single TCRB1 hybridizing band of catfish #G14 germline DNA. In contrast, 3B11 B cells show an identical unrearranged TCRB restriction pattern that matches the restriction pattern of germline DNA isolated from 3B11’s parent fish #1. The catfish 1G8 B cells also have an identical unrearranged restriction TCRB pattern. As a control for the TCRB rearrangements, the blots were hybridized with a probe for MHC class II B, a gene that would not undergo rearrangement in a cell line. As expected, each of the catfish TS32 CTL clones exhibit an MHC class II B hybridization pattern identical with that of catfish #32 genomic DNA. The hybridizing MHC class II . B patterns of G14D T cells and 3B11 B cells when compared with their respective parent fish genomic DNA show matching MHC patterns. Catfish 1G8 B cells exhibit yet a fourth MHC class II B hybridizing pattern.



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  		FIGURE 5. RT-PCR analysis of RNA encoding TCR{alpha}, TCR{beta}, and Igµ in the alloantigen-dependent CTL TS32.15 and the 3B11 B cell line. The amplified products (see Materials and Methods for primers used) were electrophoresed on a 1.0% agarose gel and stained with ethidium bromide. Catfish actin was amplified as a control. HaeIII {Phi}x174 DNA markers are shown at the left.

 


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  		FIGURE 6. Southern blot analyses showing rearrangement of the TCRB locus and MHC similarity in the catfish alloantigen-dependent CTL. EcoRI-digested genomic DNA (10 µg) from TS32.15, TS32.17, TS32.5, the cloned B cell lines 3B11 and 1G8, T cell line G14D, and erythrocytes of catfish 32, 1, and G14 hybridized with probes for catfish MHC II {beta} and TCR C{beta}. Markers (in kilobases) are shown at the left margin.

 
Catfish TCRB rearrangements

The availability of the catfish TS32 alloantigen-dependent cloned CTL lines (2) coupled with sequences of the TCRB locus from DNA isolated from catfish #32 facilitated the examination of DB and JB gene usage and junctional diversity in catfish TCRB genes. The CDR3 and framework 4 region sequences from 10 of the TS32 CTL lines and the putative TS32.4 Th cell line as well as cDNAs (termed Tb) isolated from a catfish PBL cDNA library (1) are shown in Fig. 7. Each of the cDNAs uses the single DB gene, albeit in different reading frames. Interestingly, three of the cDNAs (TS32.43, TS32.5, and Tb8) do not have a DB-encoded glycine codon. As suggested above, the CDR3 glycine residue(s) may be important for the structure of the Ag-binding CDR3 loop in TCR{beta} (24). However, it is likely that Ag binding is occurring in the TS32.43 and TS32.5 CTL, since they are both Ag specific (2, 4). A total of 13 different JB genes were used by the different cDNAs, and by comparing their rearrangements with their germline counterparts it was possible to determine N-nucleotide contributions (27) to the CDR3 regions; 11 cDNAs of 19 contained N additions. Nucleotide trimming (deletion) from the 5' and 3' ends of DB and from the 5' end of the different JB segments was also observed. No P nucleotide additions (28) were detected. The only JB group not found to be used was group VIII, which consists of the JB28 segment. Most TS32 CTL JB sequences match one of the JB germline segments. An exception is the single nucleotide difference found in the first glycine codon of the F-G-X-G motif of TS32.34, i.e., GGC is changed to a GGT. This difference may be due to either a Taq polymerase error in the 5'RACE protocol used to obtain the TS32 TCRB cDNAs or in sequencing. However, even if the nucleotide change is a bona fide difference, the codon will still encode a glycine residue. The catfish PBL Tb cDNAs also match well with the genomic JB segments. Exceptions are only found in Tb2 and Tb15; both cDNAs have two nucleotide mismatches, which may represent allelic differences, since the PBL cDNA library was derived from an outbred catfish different from fish #32. Two of the catfish cDNAs (TS32.49 and Tb8) use the allele of the JB13 segment shown as J13a in Fig. 7. In these two cDNAs, the glycine encoded by GGC at the 3' end of the CDR3 is changed to an alanine due to a single G to C nucleotide change (GCC). The TS32.49 and Tb8 V regions are spliced to the CB1*1 allele sequenced from phage {lambda}8b, and the JB13 segment sequenced from this phage contains the single G to C nucleotide change.



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  		FIGURE 7. Analysis of catfish DB and JB usage. The CDR3 and framework 4 regions of TS32 CTL and PBL (Tb) cDNAs are aligned according to J{beta} groups. The JB segment used is shown in parentheses at the right margin, and the corresponding V{beta} family is indicated beside the cDNA name. Underlines denote nucleotide changes/mismatches from genomic JB genes. TS32.49 and Tb8 use the allele of JB13, marked J13a, and nucleotide differences from JB13 are in bold. nt, nucleotide additions at the junctions; *, truncated cDNAs. Dots indicate identity, and gaps are introduced to achieve maximal alignment.

 
The TS32 CTL clones (2) appear to have a JB usage preference. Of the seven different alloantigen dependent-specific CTL, five (TS32.1, TS32.12, TS32.15, TS32.34, TS32.44) use JB members from group I, with three of them (TS32.12, TS32.15, and TS32.44) using JB 23, albeit with different junctional diversity. The other two alloantigen-specific CTL (TS32.49 and TS32.5) use group II (JB13) and group V (JB7) rearrangements, respectively. The three alloantigen-broadly-specific CTL clones use JB segments found more 5' of the CB1 gene. TS32.17 uses the group IV JB8, TS32.32 uses the group VII JB7, and TS32.43 uses the group III JB5. In contrast, the seven TCRB cDNAs isolated from the PBL library show no apparent JB bias; JB genes from groups I, II, IV, and V are used in their rearrangements (Fig. 7). Overall, seven JB segments (JB5, JB7, JB8, JB10, JB13, JB14, and JB23) belonging to five JB groups are used in successful TCRB rearrangements in the catfish TS32 CTL clones. In the putative Th cell clones TS32.4 and 28S, the group VI JB1 and group II JB15 genes are used, respectively. Four more JB segments (JB17, JB20, JB24, and JB25) representing groups I, II, IV, and V are used in the catfish PBL cDNAs. Taken together these results indicate that all catfish JB groups can probably be expressed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The channel catfish TCRB locus, like that of other vertebrates, is arranged in a translocon fashion, with a single DB gene followed by 29 JB genes and two CB genes (Fig. 8). The presence of the two CB genes in tandem is the most unusual feature of this locus. Unlike in mammals where the two TCRBC genes are either identical (22) or differ by only a few residues (21), the two catfish TCRBC genes are substantially different, with only ~36% identity at the amino acid level. This situation is reminiscent of the recently reported TCRBC genes in the bicolor damselfish, Stegastes partitus (29). In damselfish TCRB C regions are encoded by two quite divergent genes, each exhibiting significant polymorphism. In addition, C{beta} isotypic variants have been described in the Atlantic cod, Gadus morhua (30). Four different cod C{beta}s were sequenced, and one, C{beta}3, possesses a 3'UTR that shares 83% nucleotide similarity with the 3'UTRs of the predicted alleles C{beta}1 and C{beta}2, suggesting that C{beta}3 represents an isotypic variant. However, at present it is not known whether the damselfish or Atlantic cod CB genes are linked or whether they represent two different loci. Rainbow trout is the only other teleost in which TCR DB-JB-CB gene sequences have been reported. The locus consists of a single DB, followed 3' by 10 JB genes and a seemingly single CB gene (Fig. 8). Whether a second CB gene exists in trout has not been determined. The Mexican axolotl, Ambystoma mexicanum, a urodele amphibian, also appears to have multiple divergent TCRBC genes, as assessed by cDNA analyses (31, 32). Three of the axolotl cDNAs (C{beta}1, C{beta}2, and C{beta}3) share ~70% amino acid identity. The fourth (C{beta}4) has only ~42% similarity with the first three. Again, albeit genomic TCR sequences have not been cloned in the axolotl and the exact organization and structure of the TCRB locus are still unknown, cDNA analyses suggest that the TCRB locus is organized in independent clusters of DB-JB-CB and that these genes, at least in part, associate with a common pool of VBs. Here it must be emphasized that at present there are no full-length catfish TCRCB2 cDNAs. Each of the catfish T cell lines and TCR cDNAs were derived from PBL, and since preliminary RT-PCR data indicate that TCRCB2 is expressed at higher levels in intestinal lymphocytes than in PBL and other lymphoid tissues (Fig. 2), it may be that PBL are a poor source of TCRCB2-expressing cells. Even without expression data, it is still intriguing that evolutionally distinct groups of teleosts (catfish, damselfish (29), and Atlantic cod (30)) as well as axolotl (31) appear to have divergent TCR C{beta} isotypes. At present the functional significance of divergent TCR C{beta} isotypes in teleosts is unknown.



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  		FIGURE 8. Germline organization of mouse, catfish, and trout TCRB loci. Only the D, JB, and CB genes are shown, except for V{beta}14 in the mouse. *, Pseudogenes; <-, opposite transcription orientation. The schematic is to scale. Accession numbers: mouse TCR{beta}m AE000665; catfish TCR{beta}m AF410785; trout TCR{beta}, U97590.

 
The presence of 29 catfish JB genes distributed over 8.3 kb can be considered unusual. In comparison, there are 12 and 14 JB genes divided between the two DB-JB-CB clusters of human and mouse, respectively (21, 22), four in the chicken TCRB locus (33, 34), and 10 in rainbow trout (19). All of the catfish JB genes appear functional; they are in-frame and have conserved splice donor signal sequences for in-frame splicing with a CB gene (12). Each has recognizable RSS, conserved nucleotides GTG at the 3' end of the heptamer, and T/A at the fourth position of the nonamer (as described by Ramsden et al., (15)), which is considered critical for recombination in mouse recombinase substrate assays (35). The catfish JB genes can be placed into eight groups based on JB gene and RSS sequence similarity and identity and, except for group III members, are found interspersed in the germline. Yet despite the interspersion, gene duplication patterns can be discerned. The best examples being the group I and II JB10-JB19 genes, which appear to have duplicated in a series of JB gene-intron-JB gene-intron, and the tandem group III JB2-JB6 genes.

Thirteen of the catfish JB genes representing groups I–VII are used in the 19 cDNAs surveyed. Of these, 12 cDNAs were from catfish clonal T cell lines: 10 from the TS32 CTL (2) and one each from the putative Th cell lines 28S (1) and TS32.4 (2). Three of the Ag-specific CTL cDNAs (TS32.12, TS32.15, and TS32.44) use a JB23 gene rearrangement with DB in the same third reading frame, although each have different N nucleotides at the DJB junction and rearrange to different VB genes (2). However, the small survey size makes it difficult to conclusively determine whether there is JB usage preference in the catfish CTL.

In summary, genomic sequencing of catfish TCR{beta} phage clones has revealed 29 JB genes located between a single DB and two substantially different tandemly arranged CB genes. The JB genes are presumably the result of gene duplication events; all appear functional, and 13 different JB segments are found to be used in the 19 cDNAs surveyed. Catfish TCR{beta} diversity is generated by the usage of DB in all three reading frames, deletion of bases at junctions, and N nucleotide additions.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (R01AI19530) and the U.S. Department of Agriculture (99-35204-7844). Back

2 Current address: IMM-13, R218, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Back

3 Address correspondence and reprint requests to Dr. Melanie Wilson, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. E-mail: mwilson{at}microbio.umsmed.edu Back

4 Abbreviations used in this paper: CP, connecting peptide; C, constant; CYT, cytoplasmic; RSS, recombination signal sequence; TM, transmembrane; UTR, untranslated region. Back

Received for publication August 9, 2002. Accepted for publication December 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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