Opposite orientation of the alpha- and upsilon-chain constant region genes in the immunoglobulin heavy chain locus of the duck.
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Animals↗
Biomedical subjects
G W Warr
Publications and source records attributed to G W Warr.
Light chain variable region diversity in Atlantic cod (Gadus morhua L.).
This study was undertaken to determine if a lack of V(L) domain variability could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod V(L) regions was studied in 33 cDNA and two genomic clones. The variability of the CDRs was estimated by the Shannon entropy method and compared with that in other species. It was found to be lowest in the little skate (Raja erinacea), higher in cod, and highest in Xenopus and mouse. While the variability of the CDRs is slightly lower in cod than in Xenopus and mouse, it is spread over broader areas of the amino acid sequence. The length of CDR1 and CDR3 in cod is equal to or exceeds that found in skate, Xenopus, chicken and mammals. Isoelectric points and hydrophobicity vary substantially among the studied Ig light chain domains. Thus, neither the length, nor the variability, nor the physicochemical properties (pI and hydrophobicity) of the L chain CDRs can explain the absence of antibody response to immunization in cod.
Amino Acid Sequence↗
Catfish Oct2 binding affinity and functional preference for octamer motifs, and interaction with OBF-1.
The DNA-binding (POU) domain of the catfish Oct2 transcription factor was shown, by electromobility shift assays and surface plasmon resonance techniques, to have an affinity for the consensus octamer motif (ATGCAAAT) that was slightly higher than its affinity for a variant motif (ATGtAAAT). This observation is consistent with the transcriptional activation potentials of catfish Oct2 alpha and Oct2 beta, which were shown to activate transcription in catfish B and T cell lines to an equivalent extent from both the consensus and variant octamer motifs. When tested in a mouse plasmacytoma cell line, catfish Oct2 alpha and Oct2 beta, as well as mouse Oct2, showed higher transcriptional activation with the variant, as compared to the consensus, octamer motif. Catfish Oct2 was shown to function synergistically with the mammalian co-activator, OBF-1, activating octamer-dependent transcription in catfish T cells. The strong transcriptional activity of OBF-1 in catfish cells was dependent on the presence of octamer motif(s) at the proximal (promoter) rather than the distal (enhancer) position.
Animals↗
Transcriptional enhancers and the evolution of the IgH locus.
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3' Untranslated Regions↗
Characterization of Oct2 from the channel catfish: functional preference for a variant octamer motif.
The Ig heavy chain enhancer of the channel catfish (Ictalurus punctatus) has an unusual position and structure, being found in the 3' region of the mu gene and containing eight functional octamer motifs of consensus (ATGCAAAT) and variant sequences. The presence of multiple octamer motifs suggests that an Oct2 homologue may play an important role in driving expression of the Ig heavy chain locus in a teleost fish. To test this hypothesis, two catfish Oct2 cDNAs (alpha and beta) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 alpha and beta isoforms are derived by alternative RNA splicing; as determined by Southern analysis, Oct2 is a single copy gene. In comparisons with mammalian Oct2, the catfish Oct2 isoforms show high sequence conservation in their N-terminal regions and POU domains, but extensive divergence in their C-terminal regions. Catfish Oct2 a and beta are tissue restricted, bind both consensus and variant octamer motifs, and activate transcription in both catfish and murine cells. In contrast, mouse Oct2 activated transcription in mouse but not catfish cells. Catfish Oct2 beta is a more potent transcriptional activator than Oct2 alpha. In transient expression assays, catfish Oct2 beta showed a marked preference for the octamer variant, ATGtAAAT, which occurs twice in the catfish enhancer. Mouse Oct2 also showed increased activity with the variant octamer when tested in mouse B cells. Gel-shift analysis competition assays indicated that catfish Oct2 binds the consensus octamer motif with an apparently higher affinity than it does the variant motif.
Alternative Splicing↗
Secretory immune system of the duck (Anas platyrhynchos). Identification and expression of the genes encoding IgA and IgM heavy chains.
IgA has not previously been identified in waterfowl. Studies instead revealed physical and antigenic similarities between duck bile immunoglobulin (Ig) and serum IgM. Here, a differential screening approach was used to clone, from a duck spleen library, the cDNA encoding the heavy (H) chains of IgM and the Ig, identified here as IgA, occurring in duck secretions. Phylogenetic comparisons of inferred amino acid sequences of entire H chain constant (C) regions and of individual domains revealed that the duck mu chain was closest to chicken mu (54% overall identity), and duck alpha was closest to chicken alpha (50% identity). Comparison of the mu and alpha C regions revealed areas of up to 65% amino acid similarity within the C4 domains, accounting for the previously noted antigenic overlap of duck IgM and IgA. Messages for alpha and mu were detected in duck lymphoid organs but the alpha message was most abundant in the respiratory, alimentary and reproductive tracts. The alpha message first appeared around 14 days of age and reached adult levels of expression only at 35-50 days. The results indicate that the duck has a mucosal immune system which utilizes IgA; however, the delayed expression and secretion of duck IgA explains the susceptibility of ducklings to mucosal pathogens. Since the waterfowl are among the most primitive extant birds, the recognition of IgA in the duck supports the conclusion that IgA occurs throughout the class Aves and also existed in the common ancestors of birds and mammals.
Amino Acid Sequence↗
Mitogen and growth factor-induced activation of a STAT-like molecule in channel catfish lymphoid cells.
This article describes the identification of a putative STAT molecule in the channel catfish (Ictalurus punctatus), the first report of such a molecule in a 'lower' vertebrate. A monoclonal antibody against human STAT6 recognizes an approximately 100 kDa molecule that becomes activated and translocates to the nucleus upon both growth factor and mitogen stimulation of catfish leukocytes. This presumed catfish STAT binds the mammalian interferon-gamma activation site, a known motif of mammalian STAT binding, as shown by electromobility shift assays. Purification of the proteins present in these DNA complexes confirms that the catfish reactive molecule binds to the interferon-gamma activation site sequence. These results suggest that STAT molecules have been highly conserved in vertebrate evolution.
Animals↗
T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes.
Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta 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 Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta 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.
Amino Acid Sequence↗
Evolutionary variation of immunoglobulin mu heavy chain RNA processing pathways: origins, effects, and implications.
Immunoglobulins (Ig) can occur in two physical forms, soluble (secreted) and membrane bound. The soluble form is secreted from B cells, and is present in the blood and other fluids where it plays a role as an immune effector molecule. The membrane-bound form of the Ig molecule is inserted into the B-cell membrane, where it serves as a receptor for antigen. The function of the membrane-bound Ig as a receptor for antigen requires additional accessory molecules, the membrane Ig plus accessory molecules are referred to, collectively, as the B-cell receptor (BCR) complex. The secreted and membrane-bound forms of an Ig result from alternative patterns of RNA processing of the primary transcript from the heavy chain gene. IgM is the only class of Ig known to be conserved in all vertebrate species (perhaps exclusive of the agnathan fish). While the structure of the IgM heavy (mu) chain gene has been highly conserved in vertebrate evolution, the patterns of alternative RNA processing of the mu transcript show surprising diversity. In particular, the bony fish (teleosts) produce membrane mu-chain message by a splicing pathway that is quite different from that seen in other vertebrates; it results in the production of membrane IgM that lacks the C mu 4 domain. How this unusual RNA splicing pattern could have evolved and its implications for the function of the BCR in the bony fishes are considered here.
Animals↗
A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD.
IgD is considered to be a recently evolved Ig, being previously found only in primates and rodents. Here we describe, from a teleost fish (the channel catfish, Ictalurus punctatus), a novel complex chimeric Ig heavy chain, homologous, in part, to the heavy chain (delta) of IgD. In addition to alternative secretory or membrane-associated C termini, this chimeric molecule contains a rearranged variable domain, the first constant domain of mu, and seven constant domains encoded by a delta gene homolog. Identification of the catfish gene as delta is based on the following properties: sequence relatedness to mammalian delta; a location within the IgH locus that is immediately downstream of the mu gene; separate terminal exons for the secretory and membrane forms; coexpression with the complete mu chain in some but not all B cells. These results (i) suggest that IgD is an ancient immunoglobulin that was present in vertebrates ancestral to both the mammals and the ray-finned fishes, and (ii) raise the possibility that this Ig isotype may have served an as yet unidentified important function early in the evolution of the immune system.
Amino Acid Sequence↗
Functional motifs in the IgH enhancer of the channel catfish.
The transcriptional enhancer (Emu3') within the Ig heavy chain (IgH) locus of the channel catfish differs from those found in mammalian IgH loci in both its location and structure. However, upon transfection into fish or mouse lymphocytes, it activates transcription to an extent equivalent to that of the mouse IgH intronic enhancer (Emu). Potential transcription factor binding motifs in Emu3' are more numerous than in mammalian IgH enhancers, and are dispersed over 1.6 kilobases. We transfected catfish and mouse lymphoid cells with reporters under the control of artificial promoters containing motifs from the catfish enhancer. We demonstrate that 9 of 11 octamer motifs identified in the catfish enhancer, representing five variations of the consensus octamer (ATGCAAAT), are functional in both a catfish B-cell line (1B10) and the mouse plasmacytoma J558L. Only those octamer variants in which one of the first four bases is altered are active. Clear species differences in the strengths of the variant octamer motifs were evident, and in catfish B cells the ATGtAAAT motif was over threefold more active than the consensus octamer. The one muA and two muB motifs in Emu3' do not contribute to transcriptional activation. These results suggest that the relative functional contributions of IgH enhancer motifs has changed significantly during vertebrate evolution.
Animals↗
The adaptive immune system of fish.
Fish above the level of the agnatha display typical vertebrate adaptive immune responses characterized by immunoglobulins, T-cell receptors, cytokines, and major histocompatibility complex molecules. However, while the immune system of fish is quite different in its efficiency and complexity from that of higher vertebrates, our knowledge is sufficiently limited that we do not yet have a full understanding of the reasons for this difference. Filling the substantial gaps in our knowledge of the adaptive immune responses of fish will improve our understanding of their response to vaccines, and aid in the rational design of vaccines and protocols for vaccination.
Adaptation, Physiological↗
Purification of duck immunoglobulins: an evaluation of protein A and protein G affinity chromatography.
Duck serum proteins binding to protein A Sepharose CL-4B and protein G Sepharose 4 Fast Flow and eluted at pH 2.8 or 11.5 were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, radial/immunodiffusion against defined anti-immunoglobulin (Ig) reagents, and by the reactivity in immunoelectrophoresis of antisera raised in rabbits inoculated with the eluates. The results indicated that IgY (previous nomenclature 7.8S IgG) and IgY (delta Fc) (previously 5.7S IgG) bound to protein A efficiently and to protein G weakly, while IgM bound to protein A and protein G weakly. Some binding of non-Ig proteins also occurred. Attempts to separate the non-Ig proteins from the Igs by elution at different pHs (5.0, 4.0, 3.0 and 2.5) were unsuccessful, but it was found that precipitation of Igs in day-old duck serum with Na2SO4, followed by chromatography on protein A Sepharose, yielded relatively pure IgY. The efficient binding of the duck IgYs to protein A resembles high affinity binding of mammalian Igs but cannot be attributed to the Fc, as it is in mammals, since the IgY (delta Fc) does not have an Fc region. Instead, binding probably occurs through unique histidine residues occurring predominantly in the CH1 domain.
Amino Acid Sequence↗
IgY: clues to the origins of modern antibodies.
IgY is the functional equivalent of IgG in birds, reptiles and amphibia, but many aspects of its biology are poorly understood. Recent studies have increased awareness of the genetics and functions of this molecule, and have revealed its position as the ancestor of the uniquely mammalian antibodies IgG and IgE. Here, Greg Warr, Kathy Magor and David Higgins review current knowledge of IgY structure, function and expression in the context of the evolutionary role of this primitive immunoglobulin.
Amphibians↗
One gene encodes the heavy chains for three different forms of IgY in the duck.
IgY, the major Ab of the duck (Anas platyrhynchos), exists in two secreted forms and a transmembrane (TM) form. To investigate the genetic relationships of the multiple IgY we cloned the gene encoding the IgY (upsilon) heavy chains. The heavy chain of the smaller (5.7S) IgY, which lacks the third and fourth constant domains, results from the use of a unique terminal exon found in the intron between the second and third C region exons. Alternate pre-mRNA processing pathways also produce a full-length upsilon-chain and a TM form, each having four C region domains. Although the number of secretory exons and the inferred positions of intramolecular disulfide bonds indicate structural similarity between IgY and IgE, the TM exons of duck IgY share high sequence identity and a similar pattern of RNA processing with those of IgG. These results suggest that IgG and IgE may have diverged from an ancestral molecule resembling IgY.
Alternative Splicing↗
An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.
The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.
Animals↗
Development and characterization of channel catfish long term B cell lines.
The establishment of channel catfish long term cloned B cell lines, the first such cell lines from ectothermic vertebrates, is described. These diploid cell lines were developed by in vitro LPS stimulation of B cells from normal channel catfish peripheral blood in the absence of overt attempts to transform or immortalize the cells. The resultant cell lines were cloned and maintained continuously in vitro for more than 12 mo without restimulation, feeder cells, or exogenous factors. Southern blot analyses of the parental cell lines revealed multiple mu-chain gene rearrangements, suggesting a polyclonal origin for the cell lines. Additional evidence for polyclonal development was provided by the demonstration that the parental cell lines transcribed mRNA for all of the six known channel catfish VH gene families. The characterization of several clonal cell lines revealed mRNA expression for both the secreted and membrane forms of the catfish mu-chain; however, the cloned cell lines each expressed only a single VH gene and analysis of the Ig H chain locus was consistent with allelic exclusion having occurred in these cells. Flow cytometry demonstrated that the cloned and uncloned cell lines produced both cytoplasmic and cell surface IgM. This IgM contained only one of the two L chain isotypes of the channel catfish, suggesting preferential L chain usage. Although these cells did not appear morphologically to be plasma cells, they secreted moderate levels of IgM in culture. These cell lines have considerable potential for addressing questions concerning the evolution of B cell function.
Animals↗
Expression of a mouse-channel catfish chimeric IgM molecule in a mouse myeloma cell.
Fusion genes encoding a murine VH domain and the constant region domains of the mu chain from the channel catfish, Ictalurus punctatus, were stably expressed in the lambda light chain producing mouse myeloma cell line J558L. Although the pathways of pre-mRNA processing for expression of membrane (micron and secreted (microsecond) forms of the mu chain differ between mammals and teleosts, mRNAs encoding both catfish micron and microsecond were correctly expressed in the mouse myeloma cells. The mouse-channel catfish chimeric mu chain polypeptide was able to associate covalently with the mouse lambda light chain and assemble, intracellularly, into polymers of covalent structure (microL)2-8 which resembled those seen with native catfish IgM. In contrast to native catfish IgM, the mouse-catfish chimeric IgM showed the property of binding strongly to protein A of Staphylococcus aureus. The mouse-channel catfish chimeric IgM was core-glycosylated, but did not contain terminal sialic acid. Secretion rates for the chimeric IgM were low, and the possibility could not be excluded that extracellular chimeric IgM was released from dead or dying cells. The reason(s) for the intracellular retention of the chimeric IgM (probably in the endoplasmic reticulum) are not known, but those mechanisms involving retention via cysteine residues were excluded.
Animals↗