Norman W. Miller

Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus).

A tumor necrosis factor (TNF) alpha-like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNFalpha and TNFbeta homologs, overall sequence identity/similarity was slightly higher vs. TNFalpha genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNFalpha and beta genes, and supported the suggestion that TNFalpha and beta genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNFalpha mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNFalpha has been identified, and indicate that catfish TNFalpha is expressed in catfish in a manner similar to that seen in mammals.

Development and Analysis of Various Clonal Alloantigen- Dependent Cytotoxic Cell Lines from Channel Catfish

To determine the phenotypes of cytotoxic cells in channel catfish, clonal alloantigen-dependent leukocyte lines were established from mixed leukocyte cultures. Each clone was analyzed for expression of TCR α and β genes by RT-PCR and for target cell specificity by 51Cr-release assay. Based on the above criteria, the following five different cell types were identified among the 19 clones analyzed: 1) TCR αβ+ allospecific cytotoxic cells, 2) TCR αβ+ nonspecific cytotoxic cells, 3) allospecific TCR αβ+ noncytotoxic cells, 4) TCR αβ− nonspecific cytotoxic cells, and 5) TCR αβ− allospecific cytotoxic cells. The demonstration of cloned, TCR αβ+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.

T-cell receptors in channel catfish: structure and expression of TCR α and β genes

Abstract Herein are reported full length cDNA sequences for TCR α- and β-chains of the channel catfish. Included are sequences belonging to four Vα and six Vβ families which share hallmarks in common with the Vα and Vβ genes of other species. Similar to the situation in other vertebrates, the catfish Cα and Cβ sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Cα and Cβ regions are shorter than those of mammals and the catfish Cβ chain lacks a cysteine in its connecting peptide region. Two different catfish Cβ cDNA sequences were identified, suggesting the existence of either two Cβ loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cβ sequence exhibits 42% identity with the Cβ of Atlantic salmon, 41% identity with the Cβ of rainbow trout and 26% identity with Cβ of the horned shark. The catfish Cα amino acid sequence exhibits 44 and 29% identity with Cα of the rainbow trout and southern pufferfish, respectively. TCRα and β messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR α⧹β expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other lower vertebrate models.

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

The δ-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to Cμ1, seven C domain-encoding exons (δ1–δ7), and a transmembrane tail. The presence of cDNA forms showing splicing of δ7 to an exon encoding a secretory tail was interpreted to indicate that membrane (δm) and secreted (δs) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three δ 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 δ genes are highly similar in sequence and each contains a tandem duplication of δ2-δ3-δ4. 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 δs product may not be expressed as a chimeric μδ molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish δm transcripts appear to originate from IGHD1, whereas δs transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed δ gene. The third δ (IGHD2) is associated with a pseudo Cμ (IGHM2P); its presence is inferred by Southern blot analyses.

Immunoglobulin Isotypes: Structure, Function, and Genetics

Immunoglobulin (Ig) classes (in mammals, IgM, IgA, IgD, IgG, IgE) are defined by the isotypes of heavy (H) chains (µ, α, δ, γ, and e). Each isotype is in turn distinguished by unique structures in its constant region domains. These different structures confer distinctive functions on the Ig classes. When two or more Ig classes are very similar, as occurs with the four different types of IgG found in man and mouse, they are usually termed subclasses. Each isotype is encoded by a distinct gene and multiple heavy chain isoforms can be produced by alternative pathways of RNA processing, such as the secreted (slg) and membrane (mlg) forms of all H chains, or the full-length and truncated H chain isoforms of certain avian antibodies. Allelic variation in the constant (C) regions gives rise to allotypes. The different types of light (L) chains (in mammals, к and λ) are also typically referred to as isotypes. This system of classification of Igs was developed from studies of man and his immunological understudy, the mouse, and has proven useful not only in these two species, but also in other mammalian species. Although the classification of mammalian Ig classes and isotypes is quite clear, the situation with Igs from nonmammalian vertebrates is not. For example, is the shark molecule referred to as IgM really IgM? Should we call the predominant low molecular weight Ig in chickens IgG or IgY? This chapter discusses the ways in which these and similar questions have been approached.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

BackgroundThrough the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energys Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.ResultsA total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.ConclusionsThis project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Identification and expression analysis of interferon gamma genes in channel catfish

Multiple species of type I interferon (IFN) were recently identified in catfish (CF) (Ictalurus punctatus). Herein we extend these studies and report the existence of two distinct type II IFN genes in channel CF. As with zebrafish and the green spotted pufferfish, the two CF IFN-γ genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-γ2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-γ genes contain the hallmarks of authentic IFN-γ including: (1) a conserved nuclear localization site at the C terminus (CF IFN-γ2 only), (2) an IFN-γ signature sequence, (3) six putative helical regions within the mature protein, (4) one or more potential glycosylation sites, and (5) multiple mRNA instability motifs within the 3′ untranslated region. Moreover, well-characterized CF T and NK cell clones were shown to synthesize IFN-γ transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-γ and is consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production. Collectively, these studies indicate that Siluriformes possess two evolutionarily conserved IFN-γ genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.

Development of leukocyte cell lines from the channel catfish (Ictalurus punctatus)

Techniques are described for the generation of channel catfish long term leukocyte cell lines. These techniques include the isolation of peripheral blood leukocytes, purification of B cells by anti-immunoglobulin panning, mitogen stimulation, and in vitro maintenance and cloning of leukocyte cultures. Once stimulated in vitro, channel catfish leukocytes proliferate continuously without the need for exogenous growth factors or feeder cells. The various leukocyte cultures generated are heterogeneous and contain mixtures of monocyte/macrophage-like, T-like, or B cells. Clonal cell lines can be obtained from these mixed cultures by limiting dilution cloning in the presence of conditioned medium. A critical component of the culture medium is the use of channel catfish serum which is required for supporting and maintaining in vitro leukocyte proliferation.

Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus)

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

Antigen processing and presentation in teleost immune responses

Abstract Recent in vitro studies clearly demonstrate the importance of antigen processing and presentation in the generation of immune responses to T-dependent antigens (i.e. proteins and hapten-carrier conjugates) in phylogenetically lower vertebrates such as teleosts. Similar to the situation in mammals, antigens are processed and presented by accessory or antigen-presenting cells (APC), such as monocytes or macrophages, to specific lymphocytes in a seemingly alloantigen- (presumably major histocompatibility complex [MHC] or MHC-like) restricted fashion. Results show that processing involves proteolysis, which presumably occurs within acidic subcellular compartments. The requirement for processing can be circumvented by the presentation of peptide fragments of the native antigen on paraformaldehyde-fixed APC. Moreover, usage of structurally defined proteins, such as cytochrome C, as model antigens reveals that their species variants are cross-stimulatory to immune fish lymphocytes. Molecular analyses of such antigens reveal the existence of overlapping epitopes that seem to define the specificity of the immune response to such “families” of antigens but not to other unrelated (yet structurally defined) antigens. Consequently, these studies corroborate the hypothesis that immune functions in the divergent classes of vertebrates are highly conserved. Further, results from such studies also show that these immunologic processes appear to occur under low temperature regimes previously reported to suppress primary immune responses. Hence, these studies provide direct evidence that low temperature-induced immunosuppression in fish does not involve impaired APC functions. In light of the above observations indicating similarities between fish and mammalian systems, implications for fish vaccine design are also discussed.