The proliferation and clonal migration of B cells in the systemic and mucosal tissues of channel catfish suggests there is an interconnected mucosal immune system.

IgM transcripts from different mucosal and systemic tissues from a single adult channel catfish have been evaluated. Arrayed heavy chain cDNA libraries from each of these different mucosal and systemic tissues were separately constructed, hybridized with VH family specific probes and a variety of approaches were used to define their structural relationships. Baseline hybridization studies indicated that the tissue libraries had different VH expression patterns, and sequencing studies indicated this was not simply due to varying proportions of the same B cell population. In the systemic tissues of PBL, spleen, and anterior kidney >95% of the sequenced clones in the arrayed libraries represented different heavy chain rearrangements. Diversity was also found in the mucosal libraries of skin, gill lamellae, and two non-adjoining regions of the intestine, but additional populations were identified which indicated localized clonal expansion. Various clonal sets were characterized in detail, and their genealogies indicated somatic mutation accompanied localized clonal expansion with some members undergoing additional mutations and expansion after migration to different mucosal sites. PCR analyses indicated these mucosal clonal sets were more abundant within different mucosal tissues rather than in the systemic tissues. These studies indicate that the mucosal immune system in fish can express B cell transcripts differently from those found systemically. These studies further indicate that the mucosal immune system is interconnected with clonal B cells migrating between different mucosal tissues, results which yield new insight into immune diversity in early vertebrate phylogeny.

Patterns of receptor revision in the immunoglobulin heavy chains of a teleost fish.

H chain cDNA libraries were constructed from the RNA derived from seven different organs and tissues from the same individual catfish. Sequence analysis of >300 randomly selected clones identified clonal set members within the same or different tissues, and some of these represented mosaic or hybrid sequences. These hybrids expressed V(H) members of the same or different V(H) families within different regions of the same clone. Within some clonal sets multiple hybrids were identified, and some of these represented the products of sequential V(H) replacement events. Different experimental methods confirmed that hybrid clones identified in the cDNA library from one tissue could be reisolated in the cDNA pool or from the total RNA derived from the same or a different tissue, indicating that these hybrids likely represented the products of in vivo receptor revision events. Murine statistical recombination models were used to evaluate cryptic recombination signal sequences (cRSS), and significant cRSS pairs in the predicted V(H) donor and recipient were identified. These models supported the hypothesis that seamless revisions may have occurred via hybrid joint formation. The heptamers of the cRSS pairs were located at different locations within the coding region, and different events resulted in the replacement of one or both CDR as well as events that replaced the upstream untranslated region and the leader region. These studies provide phylogenetic evidence that receptor revision may occur in clonally expanded B cell lineages, which supports the hypothesis that additional levels of somatic H chain diversification may exist.

The nucleotide targets of somatic mutation and the role of selection in immunoglobulin heavy chains of a teleost fish.

Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.

Patterns of gene divergence and VL promoter activity in immunoglobulin light chain clusters of the channel catfish.

The structure and genomic organization of V, J, and C segments in multiple gene clusters of the G class of catfish light (L) chain were determined to study evolutionary patterns of cluster divergence and regulatory function. The results showed that the organizational pattern is conserved; two VL segments reside in opposite transcriptional orientation to a pseudogene JL segment (psiJ), a functional JL segment, and a CL segment. Structures within the central V-psiJ-J-C regions indicate that cluster duplication occurred after the V-V-psiJ-J-C organization became fixed. VL divergence in gene clusters subsequently occurred by mechanisms that principally targeted complementarity-determining regions. The sequence of the VL-flanking regions, which contained regulatory octamer, TATA, and Pax-5 (BSAP) binding site motifs, was conserved during G cluster duplication and within VL-flanking regions in divergent lineages of bony fish. Reporter assays of catfish B cells transfected with a 742-bp VL-flanking fragment showed promoter activity in the absence of enhancer elements. The promoter's activity doubled when coupled with the catfish IgH enhancer. A 136-bp fragment containing the motifs conserved in bony fish phylogeny and located between the leader initiation codon and the initiation sites of sterile transcripts served as a minimal promoter and provided the highest B-cell activity. These constructs, however, did not act as promoters in catfish non-lymphoid cells or mammalian BJA-B B cells or fibroblasts. These results indicate that the structure and function of VL promoter regions in the regulation and tissue specificity of L-chain gene expression evolved early in phylogeny.

Structure, genomic organization, and phylogenetic implications of six new VH families in the channel catfish.

To define members of previously unknown VH gene families, a channel catfish immunoglobulin heavy chain cDNA library was constructed and screened with probes specific for the seven known catfish VH families. Reiterative screening and sequence studies defined six new VH families, designated VH8-VH13, which brings the total number of VH families in the catfish to 13. This is the highest number of VH families presently defined in a lower vertebrate. Sequence comparisons indicate there is extensive diversity between members of different families with the greatest variability encoded within the complementarity determining regions. Genomic libraries were screened, and germline VH segments representing each of these new families were identified. The VH segments are closely linked and interspersed with members of different VH families. Each of these germline gene segments shared characteristic structural features: an upstream region that contained transcriptional regulatory elements, a leader sequence split by a short intron, an open reading frame encoding readily identified framework and complementarity determining regions, and a terminal recombination signal sequence consisting of a consensus heptamer, a 22-24 bp spacer with conserved 5'- and 3'-ends, and a consensus A-rich nonamer. Southern blot analyses estimate the number of members within these new families ranges from small (2-7 members in VH9, VH10, and VH12) to medium (9-13 members in VH8, VH11, and VH13). Thus, there are between 165 and 200 germline VH segments represented by these combined 13 families with present analyses indicating that perhaps one-half of these are pseudogenes. Phylogenetic comparisons indicate that members of these different catfish VH families cluster within Groups C and D of vertebrate VH genes. These analyses also indicate that Group D is represented by two different branches and both branches include VH families from different lineages of bony fish that diverged early in vertebrate phylogeny.

Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters.

Two structurally-related genomic clusters of catfish immunoglobulin heavy chain gene segments are known. The first gene cluster contains DH and JH segments, as well as the C region exons encoding the functional Cmu. The second gene cluster contains multiple VH gene segments representing different VH families, a germline-joined VDJ, a single JH segment, and at least two pseudogene Cmu exons. It was not known whether these gene clusters were linked, nor was the organization or the location of VH segments associated within the first gene cluster known. Pulsed-field gel electrophoresis studies have been used to determine the structural organization of these gene clusters. Restriction mapping studies show that the two gene clusters are closely linked; the second gene cluster is located upstream from the first with the Cmu regions within the clusters separated by about 725kb. The clusters are in the same relative transcriptional orientation, and the results indicate that the complete IgH locus spans no more than 1000kb and may be as small as 750-800kb. VH gene segments are located both upstream and downstream of the pseudo-Cmu exons; however, no VH gene segments that hybridized with the VH specific probes were detected downstream of the functional Cmu. These studies coupled with earlier sequence analyses indicate that the catfish IgH locus arose from a massive internal duplication event. Subsequent gene rearrangement within the duplicated cluster likely resulted in the presence of the germline VDJ and the deletion of intervening V, D and J segments. Transposition by a member of the Tc1/mariner family of transposable elements appears to have led to the disruption of the duplicated Cmu.