Kazuhiro Fujiki

Analysis of genomic and expressed major histocompatibility class Ia and class II genes in a hexaploid Lake Tana African ‘large’ barb individual (Barbus intermedius)

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC classxa0I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some classxa0IIB loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed classxa0II MHC genes in a hexaploid African ‘large’ barb. This prompted us to study the number of MHC genes present in the genome of an African ‘large’ barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic classxa0Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of classxa0I and IIB genes. Our study revealed three β2-microglobulin, five classxa0Ia, four classxa0IIA, and four classxa0IIB genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The classxa0Ia and classxa0II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.

Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.

Molecular cloning and characterization of calreticulin from rainbow trout ( Oncorhynchus mykiss).

Calreticulin (CRT) is a highly conserved, high-capacity, calcium-binding protein shared among vertebrates, invertebrates and higher plants. Its biological importance, highlighted by its highly conserved nature, is supported by its crucial physiological and immunological functions. Within the endoplasmic reticulum, CRT serves as a calcium modulator and a lectin-like chaperone for glycoproteins, especially class I major histocompatibility receptors. To date, CRT cDNA clones have been isolated from a wide range of phyla, yet little is known about this gene in fish species, the largest and most diverse group of jawed vertebrates. This report describes the cloning of a cDNA from a rainbow trout pronephros library that encodes a deduced 419-amino acid protein, which includes a predicted 20-amino acid signal peptide and has a 69% amino acid identity to both murine and human CRT. Like its mammalian counterparts, this cDNA contains conserved cysteine residues believed to form a disulphide bond, a proline-rich region which includes a potential N-glycosylation site, and a highly acidic C-terminal domain terminating with the endoplasmic reticulum retrieval sequence, KDEL. Reverse transcription tissue-distribution assays indicate it is ubiquitously expressed in all tissues tested with highest expression in liver, while Southern blotting indicates it is a single copy gene.

Molecular cloning and characterisation of a carp (Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF ☆

In the course of suppression subtractive hybridisation between sodium alginate-induced peritoneal cells (SA-PC) and normal head kidney cDNAs in common carp (Cyprinus carpio), a cytokine-like cDNA clone was found. The clone, named M17, contains a 1600bp nucleotide sequence that encodes a 215 amino acid putative protein that would have a pI of 9.01 and would include a 33 amino acid signal peptide. The 3 untranslated region has seven ATTTA mRNA destabilising motifs that are common in cytokines and oncogenes. In a BLASTP search, M17 was most similar to chicken ciliary neurotrophic factor (CNTF) with 25% amino acid identity, followed by mammalian CNTF, cardiotrophin-1 and leukemia inhibitory factor (LIF) all of which belong to the IL-6 subfamily. However, M17 has some differences with CNTF in that CNTF has no signal sequence, the gene organisation of M17 is three exons and two introns, whereas that of CNTF is two exons and one intron, M17 has seven cysteines while CNTF has one cysteine, and M17 mRNA is detected in peripheral blood leukocytes as well as brain, whereas CNTF is expressed only in the nervous system. Compared to other members in the IL-6 subfamily cytokines, M17s cysteine positions and gene organisation are similar to those of oncostatin M and LIF, although amino acid identities are only 15-17%. Southern hybridisation suggested that M17 is a single copy gene. SA-PC showed significantly higher M17 mRNA levels than normal head kidney cells, which are considered to be a source of the SA-PC, indicating that M17 is inducible by inflammatory stimulation.

Lens and retina formation require expression of Pitx3 in Xenopus pre-lens ectoderm

Pitx3 is expressed in tissues fated to contribute to eye development, namely, neurula stage ectoderm and prechordal mesoderm, then presumptive lens ectoderm, placode, and finally lens. Pitx3 overexpression alters lens, optic cup, optic nerve, and diencephalon development. Many of the induced anomalies are attributable to midline deficits; however, as assessed by molecular markers, ectopic Pitx3 appears to temporarily enlarge the lens field. These changes are usually insufficient to generate either ectopic lenses to enlarge the eye that eventually differentiates. Conversely, use of a repressor chimera or of antisense morpholinos alters early expression of marker genes, and later inhibits lens development, thereby abrogating retinal induction. Reciprocal grafting experiments using wild‐type and morpholino‐treated tissues demonstrate that Pitx3 expression in the presumptive lens ectoderm is required for lens formation. Contradictory to recent assertions that retina can form in the absence of a lens, the expression of Pitx3 in the presumptive lens ectoderm is critical for retina development. Developmental Dynamics 234:577–589, 2005.

Genomic cloning of novel isotypes of the rainbow trout interleukin-8

A cDNA clone, designated IL-8nL, was obtained by suppression subtractive hybridisation between lipopolysaccharide-stimulated and non-stimulated populations of the rainbow trout macrophage-like cell line, RTS11. IL-8nL was similar but not identical to a recently published sequence of the gene encoding rainbow trout interleukin-8 (IL-8). Amplification of genomic DNA by the polymerase chain reaction (genomic PCR) using a single outbred trout with common primers in the 5′ and 3′ untranslated regions gave six distinct genomic sequences, including one (IL-8A) almost identical to that of the published IL-8 gene and another identical to IL-8nL. The other four clones were termed IL-8B, IL-8C, IL-8D and IL-8E. The deduced amino acid sequences of IL-8A through IL-8E are all identical to the published IL-8, while the IL-8nL protein has a substitution of Arg87 to Lys. Analysis of ten outbred trout by genomic PCR of a repeat region in exon 4, which has three different sizes in the above alleles, revealed a shorter, fourth fragment termed IL-8X and another of the same size as IL-8nL, but with a different single nucleotide replacement, called IL-8nL2. These results, together with a Southern blot of the same ten individuals showing up to five bands, indicate that rainbow trout has at least four copies of the IL-8 gene. Like IL-8nL, IL-8X lacks the repeat sequence in exon 4 and encodes a protein identical to IL-8nL protein. Polymerase chain reaction of the repeat region was useful for typing rainbow trout into four categories, and the type III and IV fish have a new allele, IL-8F, which lacks one repeat unit compared with IL-8A.

Cloning and characterization of cDNA clones encoding CD9 from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Abstract. In comparison to mammals, relatively few of the molecules involved in teleost immune responses have been isolated and characterized. A rapid method of isolating molecules important for immune function is subtractive hybridization. One such experiment using infectious hematopoietic necrosis virus-infected Atlantic salmon produced several cDNA clones with similarity to mammalian immune-specific genes, including granzyme M (accession no. AF434669) and CD9. After cloning the rainbow trout version of CD9, sequence analysis showed that both salmonid sequences contained many features of the tetraspanin receptor family to which CD9 belongs. Phylogenetic analysis revealed a close association of the trout and salmon sequences to known CD9 and CD81 receptors. Southern blotting demonstrated that the rainbow trout gene is single copy. Reverse transcriptase PCR showed strong expression of this clone in many tissues, but liver expression was very low – an observation consistent with the clone being a CD9, not a CD81, equivalent. The evidence suggests that the sequences reported here are bona fide teleost CD9 homologues and we are currently producing recombinant proteins and polyclonal antisera for use in functional studies.

A Cell Line (HEW) from Embryos of Haddock (Melanogrammus aeglefinius) and Its Capacity to Tolerate Environmental Extremes

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitzs L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18°C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28°C or being switch to a simple salt solution (SSS). At 28°C, cells died within 24xa0h. In SSS, HEW cells survived as a monolayer for at least 7xa0days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.

Alternate forms of MHC class II-associated invariant chain are not produced by alternative splicing in rainbow trout (Oncorhynchus mykiss) but are encoded by separate genes.

A major limiting factor in understanding teleost major histocompatibility receptor function is the lack of knowledge about antigen presentation accessory molecules. We report here two cDNA clones encoding teleost versions of invariant chain and one encoding a related protein that may play a protease inhibition role in antigen presentation. The two invariant chain equivalents are similar to each other where they overlap, but differ in the presence or absence of a thyroglobulin domain. This domain is added to tetrapod invariant chain protein by alternative splicing but there was no evidence of alternative splicing of the two trout genes. Southern blotting confirmed that all three trout cDNAs are derived from single copy genes and Northern blotting indicated that they are expressed in antigen tissues. Thus the encoded proteins are probably involved in antigen presentation, but their expression is probably regulated in a manner different from tetrapods.

Cloning and characterization of cDNA clones encoding membrane-bound and potentially secreted major histocompatibility class I receptors from walleye (Stizostedion vitreum)

Abstract. Major histocompatibility (MH) gene polymorphism has been used to type populations of humans, mice, and fish. Walleye (Stizostedion vitreum) comprise an economically important fishery in Lake Erie, but whether those in the western basin form a single population or separate shoal- and river-breeding populations is not known. To develop MH gene markers for use in defining their population structure, we constructed a head kidney cDNA library from which five full-length classxa0I heavy-chain clones were isolated and sequenced. Although they came in roughly three sizes, 1300, 1400, and over 2000xa0bp, the clones all exhibited a high degree of sequence similarity to each other and to known teleost MH classxa0I cDNAs in the area encoding the extracellular domains, but showed dramatic differences in their transmembrane and cytoplasmic domains. One clone had an AG repeat that eliminated the hydrophobicity of the transmembrane domain, indicating that it may encode a secreted classxa0I receptor. The other four clones encode three distinctly different cytoplasmic domains. The two clones that encode the same cytoplasmic domain resemble those of the known teleost MH classxa0I sequences the most. Southern blotting indicated that there were four copies of the gene present in the walleye genome. Northern blotting showed that classxa0I MH genes are expressed in most tissues and mRNAs of all three size classes can be detected. A preliminary survey of the polymorphism of these genes indicates that they will provide useful markers for differentiating fish stocks.