Thomas J. McConnell

MHC class I genes of the channel catfish: sequence analysis and expression

Abstract Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β2-microglobulin (β2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I α) was associated with β2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β2m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II A GENE POLYMORPHISM IN THE STRIPED BASS

Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex(Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating in the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the α1 encoding domain. The encoded α2, transmembrane, and cytoplasmic regions of all seven striped genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass.

Angle-resolved Mueller matrix study of light scattering by B-cells at three wavelengths of 442, 633, and 850 nm

Angle-resolved signals of polarized light scattered by biological cells provide rich information on cell morphology. Quantitative study of these signals can lead to new methods to develop and improve high-throughput instrumentation for cell probing such as scattering-based flow cytometry. We employ a goniometer system with a photoelastic modulation scheme to determine selected Mueller matrix elements of B-cell hydrosol samples. The angular dependence of S(11), S(12), and S(34) is determined from the scattered light signals between 10 and 160 deg at the three wavelengths 442, 633, and 850 nm. A finite-difference, time-domain (FDTD) method and coated-sphere model are used to investigate the effect of nuclear refractive index on the angle-resolved Mueller elements at different wavelengths using the 3-D structures of selected B cells reconstructed from confocal images. With these results, we demonstrate the value of the light-scattering method in obtaining the cell morphology information.

Variability in an MHCMosa class II β chain-encoding gene in striped bass (Morone saxatilis)

The major histocompatibility complex (MHC) class II B locus of the striped bass (Morone saxatilis) was found to contain multiple forms of the class II B gene. Seven complete MHC class II B cDNA clones were isolated and sequenced, identifying five unique allelic forms of a MHC class II B gene. Among three specimens, each representing a geographically distinct population (Chesapeake Bay, MD; Roanoke River, NC; and Santee-Cooper Reservoir, SC) extensive variability was detected in the beta 1 encoding domain, which corresponds with the functional peptide-binding region (PBR) of known MHC class II molecules. The location of variable amino acid residues in the beta 1 domains corresponds with polymorphic sites observed in other teleosts and higher vertebrates. The amino acid translated beta 2 domain encoding regions, transmembrane regions, and cytoplasmic regions of the five clones correlated well with those of known vertebrate MHC class II proteins. Seventy-one percent of the variability found within the presumed PBR encoded at the MHCMosa class II B locus corresponded with that of the PBR of a human MHC class II B gene. Overall, the Mosa sequences showed greatest similarity to the MHC class II B genes of cichlid fishes, as expected from phylogenetic relationships.

Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus).

Abstract: Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II β1 domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts.

Expressed major histocompatibility complex class II loci in fishes

Summary: Peptides derived from parasites are presented to T helper cells by major histocompatibility complex (MHC) class II αβ heterotrimeric cell‐surface molecules. In mice and humans, the genes encoding these antigen‐presenting molecules are known to be polymorphic and poly‐genic. Multiple loci for MHC class II A and E genes are proposed to allow for an increased peptide‐binding repertoire. The multigenic nature of expressed MHC class II loci and the differences between these loci in fishes are the focus of this review, Particular emphasis is placed on an evolutionary comparison of class II B loci, especially two class 11 B loci that have undergone dramatic changes from one another suggesting an ancestral gene duplication event that took place at an early stage in the evolution of teleosts, The number of functional class II αβ loci heterotrimers may have a profound impact on the organisms ability to battle constantly evolving parasitic infections.

Immunologic characterization of the recombinant fire ant venom allergen Sol i 3.

Individuals sensitized to fire ant stings show immunoglobulin (Ig)E antibodies against the venom protein Sol i 3. We determined the full‐length complementary DNA (cDNA) sequence of this protein and expressed recombinant Sol i 3 in immunogenic form. The complete cDNA of Sol i 3 was obtained by reverse transcription polymerase chain reaction (RT‐PCR) and PCR + 1 reactions using gene‐specific oligonucleotides, and oligonucleotides designed from the amino acid sequence of this protein. The encoding cDNA is 705 bp in length corresponding to 235 amino acids. The first 22 amino acids are a leader sequence. The protein with an added C‐terminal hexahistidine tag was expressed in insect cells using a baculovirus system. The recombinant protein was secreted into the supernatant and affinity purified with a cobalt chelating resin. The recombinant fire ant venom allergen Sol i 3 showed similar IgE binding activity to the native protein in radioallergosorbent test (RAST) and RAST inhibition assays. It was produced in both a glycosylated and an unglycosylated form. A three‐dimensional reconstruction of Sol i 3 was compared with the experimentally determined structure of the related allergen Ves v 5. This model is supported by results of circular dichroism spectroscopy.

Production of a recombinant imported fire ant venom allergen, Sol i 2, in native and immunoreactive form

BACKGROUND The complementary DNA encoding for the important imported fire ant venom allergen, Sol i 2, has previously been cloned. The binding of human IgE antibodies to Sol i 2 has been demonstrated to be conformation-dependent. METHODS A couple cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinant Sol i 2 was isolated and characterized. RESULTS Recombinant (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 micrograms/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was identical to that of the natural molecule. No significant increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal antibody was lost by heating in sodium dodecylsulfate and reduction. CONCLUSION A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.

IL-4 responsive CD4+ T cells specific for myelin basic protein: IL-2 confers a prolonged postactivation refractory phase

This study compared myelin basic protein‐specific T cells from Lewis rats that were derived in the presence of either rat IL‐4 or IL‐2. Interleukin‐4 was a maintenance factor that enabled derivation of long‐term T cell lines. When activated, IL‐4 dependent lines were lacking in IL‐2 production capacity but maintained high levels of responsiveness to IL‐2 and recognized IL‐2 as a dominant growth factor. Activated IL‐4 dependent T cells rapidly reverted to a quiescent phenotype in the presence of IL‐4 and rapidly regained myelin basic protein reactivity. In contrast, activated IL‐2 dependent T cells that were propagated in IL‐2 had a more persistent blastogenic phenotype and a prolonged refractory phase. Interleukin‐4 dependent lines that were propagated in IL‐2 up‐regulated the capacity to produce IL‐2 and also acquired prolonged postactivation refractoriness. Thus, IL‐2 was a dominant growth factor that conferred prolonged activation‐dependent non‐responsiveness. The coupling of dominant growth factor activity with prolonged postactivation refractoriness may be associated with the requisite role of IL‐2 in homeostatic self‐tolerance.

Nucleotide sequence of cDNA encoding the fire ant venom protein Sol i II

For the first time the cDNA encoding a fire ant venom protein has been sequenced. Oligonucleotides were designed according to the amino acid sequence. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by the PCR technique. Comparison to the amino acid sequence of the venom protein shows a leader sequence 19 amino acids long.