An FcR homolog (IpFcRI), representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish (Ictalurus punctatus). Mining of the catfish expressed sequence tag databases using mammalian FcR sequences for CD16, CD32, and CD64 resulted in the identification of a teleost Ig-binding receptor. IpFcRI is encoded by a single-copy gene containing three Ig C2-like domains, but lacking a transmembrane segment and cytoplasmic tail. The encoded Ig domains of IpFcRI are phylogenetically and structurally related to mammalian FcR and the presence of a putative Fc-binding region appears to be conserved. IpFcRI-related genomic sequences are also present in both pufferfish and rainbow trout, indicating the likely presence of a soluble FcR in other fish species. Northern blot and qualitative PCR analyses demonstrated that IpFcRI is primarily expressed in IgM-negative leukocytes derived from the lymphoid kidney tissues and PBL. Significantly lower levels of IpFcRI expression were detected in catfish clonal leukocyte cell lines. Using the native leader, IpFcRI was secreted when transfected into insect cells and importantly the native IpFcRI glycoprotein was detected in catfish plasma using a polyclonal Ab. Recombinant IpFcRI binds catfish IgM as assessed by both coimmunoprecipation and cell transfection studies and it is presumed that it functions as a secreted FcR akin to the soluble FcR found in mammals. The identification of an FcR homolog in an ectothermic vertebrate is an important first step toward understanding the evolutionary history and functional importance of vertebrate Ig-binding receptors.

Previously a cDNA encoding a putative interferon gene, designated CF IFN-1, was identified from a catfish EST library. However, its constitutive expression, absence of a signal peptide, and apparently low level of biological activity suggested that this cDNA likely encoded an expressed pseudogene. Since Southern blot analysis suggested the presence of two to three IFN genes, additional cDNAs were generated from catfish fibroblast and lymphoid cell lines using primers designed to conserved regions of zebrafish and catfish interferon. Using this approach, three novel CF IFN genes, two of which likely encode functional interferon molecules, were identified. At the amino acid level, similarity among CF IFNs ranged from 71% to 82%, whereas similarity to other fish IFNs ranged from 15% to 35%. Although CF IFN-3, like CF IFN-1, lacks a signal peptide, CF IFN-2 and -4 appear to encode full-length, signal sequence-bearing genes. Consistent with their putative identification as functional genes, CF IFN-2 and -4 were not expressed in unstimulated cell lines, and CF IFN-2 was rapidly upregulated in CCO cells in response to virus infection or treatment with dsRNA. Moreover, as with salmon, fugu, and zebrafish interferon genes, CF IFN-1 contained four introns whose locations were conserved not only with respect to other fish IFNs, but also with respect to mammalian IFN-lambda. While it is likely that CF IFNs represent Type I IFNs, several characteristics preclude assigning these cytokines to any particular subfamily.

The channel catfish, Ictalurus punctatus, is widely recognized as an important model for studying immune responses in ectothermic vertebrates. It is one of the few fish species for which defined viable in vitro culture systems have been established and is currently the only fish species from which a variety of functionally distinct clonal leukocyte lines are available. Moreover, there is a large basis of biochemical and molecular information on the structure and function of catfish immunoglobulins (Igs). Catfish, as other teleosts, have a tetrameric homolog of IgM as their predominant serum Ig plus a homolog of IgD. They also have genetic elements basically similar to those of mammals, which encode and regulate their expression. The catfish Ig heavy (H) chain locus is a translocon-type locus with three Igdelta genes linked to an Igmu gene or pseudogene. The catfish IgH locus is estimated to contain approximately 200 variable (V) region genes representing 13 families as well as at least three diversity (D) and 11 joining (JH) genes. The catfish has two light (L) chain isotypes, F and G, both encoded by loci organized in multiple cassettes of VL-JL-CL with the VL in the opposite transcriptional orientation. Hence, all requisite components for encoding antibodies are present in the catfish, albeit with certain variations. In the future, whether or not additional unique features of Ig function and expression will be found remains to be determined.

The Oct2 transcription factor is important in driving expression of the IgH locus of the channel catfish, Ictalurus punctatus. Two isoforms, catfish Oct2alpha and Oct2beta, have been characterized at the level of expression and function, but little is known of the structure of the Oct2 gene in catfish. To gain insight into the diversity of Oct2 gene structure and expression in the teleost fish, a comparative genomic analysis of Oct2 was undertaken in the pufferfish (Fugu rubripes) and the zebrafish (Danio rerio). The orthologues of zebrafish and Fugu Oct2 were identified, and share with catfish Oct2 the expression of a limited number (two in zebrafish, three in Fugu) of isotypes produced by alternative pathways of RNA processing. The alternatively spliced variants of catfish Oct2 showed a different pattern of exon use from those of Fugu and zebrafish. The analysis also identified a novel homologue of Oct2 in both zebrafish and Fugu. This homologue, termed Oct2x, shares similarities to both Oct1 and Oct2. A phylogenetic analysis of the relationships of Oct2x gave an unexpected result, with Oct2x occupying a position basal to the Oct gene families of both vertebrates and Drosophila.

The function of the transcriptional enhancer, Emu3', of the IgH locus of the channel catfish, Ictalurus punctatus, involves the interaction of E-protein and Oct family transcription factors. The E-proteins [class I basic helix-loop-helix (bHLH) family] are encoded in mammals by three genes: E2A (of which E12/E47 are alternatively spliced products), HEB, and E2-2. An E2A homologue has been identified in a catfish B-cell cDNA library and contains regions homologous to the bHLH and activation domains of mammalian and other vertebrate E2A proteins. E2A message is widely expressed, being readily detected in catfish B cells, T cells, kidney, spleen, brain, and muscle. Its expression is lower than that previously observed for TF12/CFEB, the catfish homologue of HEB. E2A strongly activated transcription of a muE5 motif-dependent construct in catfish B cells, and also activated transcription from the core region of the catfish IgH enhancer (Emu3') in a manner dependent on the presence of the muE5 site. Catfish E2A, expressed in vitro, bound the muE5 motif present in the core region of Emu3'. These results document the conservation of structure and function in vertebrate E2A and suggest a potential role of E2A in driving expression of the IgH locus at the phylogenetic level of a teleost fish.

E proteins are essential for B lymphocyte development and function, including immunoglobulin (Ig) gene rearrangement and expression. Previous studies of B cells in the channel catfish (Ictalurus punctatus) identified E protein homologs that are capable of binding the muE5 motif and driving a strong transcriptional response. There are three E protein genes in mammals, HEB (TCF12), E2A (TCF3), and E2-2 (TCF4). The major expressed E proteins found in catfish B cells are homologs of HEB and of E2A. Here we sought to define the complete family of E protein genes in a teleost fish, Takifugu rubripes, taking advantage of the completed genome sequence. The catfish CFEB (HEB homolog) sequence identified homologous E-protein-encoding sequences in five scaffolds in the Takifugu genome database. Detailed comparative analysis with the human genome revealed the presence of five E protein homologs in Takifugu. Single genes orthologous to HEB and to E2-2 were identified. In contrast, two members of the E2A gene family were identified in Takifugu; one of these shows the alternative processing of transcripts that identifies it as the ortholog of the E12/E47-encoding mammalian E2A gene, whereas the second Takifugu E2A gene has no predicted alternative splice products. A novel fifth E protein gene (EX) was identified in Takifugu. Phylogenetic analysis revealed four E protein branches among vertebrates: EX, E2A, HEB, and E2-2.

Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.

The transcriptional enhancer (Emu3') of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single muE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a muE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Emu3') in a manner dependent on the presence of the muE5 site. Both CFEB1 and -2 bound the muE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

To elucidate cytolytic mechanisms in the channel catfish, lysates from catfish lymphoid and fibroblast cell lines were screened by Western blot analysis using a panel of antibodies reactive with components of the mammalian apoptotic pathway. Strong reactivity with three proteins (approximate Mr 70,000, 37,000, and 15,000) was seen using an antibody targeted to mammalian Fas ligand (FasL). The sizes of the two smaller proteins are consistent with their tentative designation as membrane-bound (37,000 Mr) and soluble (15,000 Mr) FasL. Treatments known to induce FasL in mammalian systems (e.g., PMA/calcium ionophore, UV-irradiation) induced expression of the 37,000- Mr protein in catfish T-cell lines. Moreover, expression of the 37,000- Mr protein in clonal T cells was up-regulated by increasing cell density. At the nucleotide level, homologues of Fas receptor (FasR), FADD, and caspase 8 were identified and characterized. These gene products likely constitute the teleost equivalent of the death-inducing signaling complex (DISC). FADD was constitutively expressed in all (T, B, macrophage, and fibroblast) cell lines examined as well as in peripheral blood lymphocytes (PBL), whereas FasR and caspase 8 were expressed in all cell lines except CCO, a FasL-positive fibroblast line. In contrast to FasL, expression of FasR and caspase 8 was inversely proportional to cell density. Collectively these studies identified four membrane-proximal proteins involved in the initiation of apoptosis in channel catfish and suggest that mechanisms of cell-mediated cytotoxicity in teleosts are similar to those used by mammals.