Tetraploid Ancestry Provided Atlantic Salmon With Two Paralogue Functional T Cell Receptor Beta Regions Whereof One Is Completely Novel.

Protective cellular immune responses have been difficult to study in fish, due to lack of basic understanding of their T cell populations, and tools to study them. Cellular immunity is thus mostly ignored in vaccination and infection studies compared to humoral responses. High throughput sequencing, as well as access to well assembled genomes, now advances studies of cellular responses. Here we have used such resources to describe organization of T cell receptor beta genes in Atlantic salmon. Salmonids experienced a unique whole genome duplication approximately 94 million years ago, which provided these species with many functional duplicate genes, where some duplicates have evolved new functions or sub-functions of the original gene copy. This is also the case for T cell receptor beta, where Atlantic salmon has retained two paralogue T cell receptor beta regions on chromosomes 01 and 09. Compared to catfish and zebrafish, the genomic organization in both regions is unique, each chromosomal region organized with dual variable- diversity- joining- constant genes in a head to head orientation. Sequence identity of the chromosomal constant sequences between TRB01 and TRB09 is suggestive of rapid diversification, with only 67 percent as opposed to the average 82-90 percent for other duplicated genes. Using virus challenged samples we find both regions expressing bona fide functional T cell receptor beta molecules. Adding the 292 variable T cell receptor alpha genes to the 100 variable TRB genes from 14 subgroups, Atlantic salmon has one of the most diverse T cell receptor alpha beta repertoire of any vertebrate studied so far. Perhaps salmonid cellular immunity is more advanced than we have imagined.

Fate of MHCII in salmonids following 4WGD.

Major histocompatibility complex (MHC) genes are key players in the adaptive immunity providing a defense against invading pathogens. Although the basic structures are similar when comparing mammalian and teleost MHC class II (MHCII) molecules, there are also clear-cut differences. Based on structural requirements, the teleosts non-classical MHCII molecules do not comply with a function similar to the human HLA-DM and HLA-DO, i.e., assisting in peptide loading and editing of classical MHCII molecules. We have previously studied the evolution of teleost class II genes identifying various lineages and tracing their phylogenetic occurrence back to ancient ray-finned fishes. We found no syntenic MHCII regions shared between cyprinids, salmonids, and neoteleosts, suggesting regional instabilities. Salmonids have experienced a unique whole genome duplication 94 million years ago, providing them with the opportunity to experiment with gene duplicates. Many salmonid genomes have recently become available, and here we set out to investigate how MHCII has evolved in salmonids using Northern pike as a diploid sister phyla, that split from the salmonid lineage prior to the fourth whole genome duplication (4WGD) event. We identified 120 MHCII genes in pike and salmonids, ranging from 11 to 20 genes per species analyzed where DB-group genes had the most expansions. Comparing the MHC of Northern pike with that of Atlantic salmon and other salmonids species provides a tale of gene loss, translocations, and genome rearrangements.

A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples.

BACKGROUND: Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012-2013 on more than 2000 faecal samples. METHODS: We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. RESULTS: Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7-100). CONCLUSIONS: The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection.

BACKGROUND: Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. FINDINGS: Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. CONCLUSIONS: The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

Comprehensive analysis of MHC class I genes from the U-, S-, and Z-lineages in Atlantic salmon.

BACKGROUND: We have previously sequenced more than 500 kb of the duplicated MHC class I regions in Atlantic salmon. In the IA region we identified the loci for the MHC class I gene Sasa-UBA in addition to a soluble MHC class I molecule, Sasa-ULA. A pseudolocus for Sasa-UCA was identified in the nonclassical IB region. Both regions contained genes for antigen presentation, as wells as orthologues to other genes residing in the human MHC region. RESULTS: The genomic localisation of two MHC class I lineages (Z and S) has been resolved. 7 BACs were sequenced using a combination of standard Sanger and 454 sequencing. The new sequence data extended the IA region with 150 kb identifying the location of one Z-lineage locus, ZAA. The IB region was extended with 350 kb including three new Z-lineage loci, ZBA, ZCA and ZDA in addition to a UGA locus. An allelic version of the IB region contained a functional UDA locus in addition to the UCA pseudolocus. Additionally a BAC harbouring two MHC class I genes (UHA) was placed on linkage group 14, while a BAC containing the S-lineage locus SAA (previously known as UAA) was placed on LG10. Gene expression studies showed limited expression range for all class I genes with exception of UBA being dominantly expressed in gut, spleen and gills, and ZAA with high expression in blood. CONCLUSION: Here we describe the genomic organization of MHC class I loci from the U-, Z-, and S-lineages in Atlantic salmon. Nine of the described class I genes are located in the extension of the duplicated IA and IB regions, while three class I genes are found on two separate linkage groups. The gene organization of the two regions indicates that the IB region is evolving at a different pace than the IA region. Expression profiling, polymorphic content, peptide binding properties and phylogenetic relationship show that Atlantic salmon has only one MHC class Ia gene (UBA), in addition to a multitude of nonclassical MHC class I genes from the U-, S- and Z-lineages.

Multiple expressed MHC class II loci in salmonids; details of one non-classical region in Atlantic salmon (Salmo salar).

BACKGROUND: In teleosts, the Major Histocompatibility Complex (MHC) class I and class II molecules reside on different linkage groups as opposed to tetrapods and shark, where the class I and class II genes reside in one genomic region. Several teleost MHC class I regions have been sequenced and show varying number of class I genes. Salmonids have one major expressed MHC class I locus (UBA) in addition to varying numbers of non-classical genes. Two other more distant lineages are also identifyed denoted L and ZE. For class II, only one major expressed class II alpha (DAA) and beta (DAB) gene has been identified in salmonids so far. RESULTS: We sequenced a genomic region of 211 kb encompassing divergent MHC class II alpha (Sasa-DBA) and beta (Sasa-DBB) genes in addition to NRGN, TIPRL, TBCEL and TECTA. The region was not linked to the classical class II genes and had some synteny to genomic regions from other teleosts. Two additional divergent and expressed class II sequences denoted DCA and DDA were also identified in both salmon and trout. Expression patterns and lack of polymorphism make these genes non-classical class II analogues. Sasa-DBB, Sasa-DCA and Sasa-DDA had highest expression levels in liver, hindgut and spleen respectively, suggestive of distinctive functions in these tissues. Phylogenetic studies revealed more yet undescribed divergent expressed MHC class II molecules also in other teleosts. CONCLUSION: We have characterised one genomic region containing expressed non-classical MHC class II genes in addition to four other genes not involved in immune function. Salmonids contain at least two expressed MHC class II beta genes and four expressed MHC class II alpha genes with properties suggestive of new functions for MHC class II in vertebrates. Collectively, our data suggest that the class II is worthy of more elaborate studies also in other teleost species.

Genomic organization of duplicated major histocompatibility complex class I regions in Atlantic salmon (Salmo salar).

BACKGROUND: We have previously identified associations between major histocompatibility complex (MHC) class I and resistance towards bacterial and viral pathogens in Atlantic salmon. To evaluate if only MHC or also closely linked genes contributed to the observed resistance we ventured into sequencing of the duplicated MHC class I regions of Atlantic salmon. RESULTS: Nine BACs covering more than 500 kb of the two duplicated MHC class I regions of Atlantic salmon were sequenced and the gene organizations characterized. Both regions contained the proteasome components PSMB8, PSMB9, PSMB9-like and PSMB10 in addition to the transporter for antigen processing TAP2, as well as genes for KIFC1, ZBTB22, DAXX, TAPBP, BRD2, COL11A2, RXRB and SLC39A7. The IA region contained the recently reported MHC class I Sasa-ULA locus residing approximately 50 kb upstream of the major Sasa-UBA locus. The duplicated class IB region contained an MHC class I locus resembling the rainbow trout UCA locus, but although transcribed it was a pseudogene. No other MHC class I-like genes were detected in the two duplicated regions. Two allelic BACs spanning the UBA locus had 99.2% identity over 125 kb, while the IA region showed 82.5% identity over 136 kb to the IB region. The Atlantic salmon IB region had an insert of 220 kb in comparison to the IA region containing three chitin synthase genes. CONCLUSION: We have characterized the gene organization of more than 500 kb of the two duplicated MHC class I regions in Atlantic salmon. Although Atlantic salmon and rainbow trout are closely related, the gene organization of their IB region has undergone extensive gene rearrangements. The Atlantic salmon has only one class I UCA pseudogene in the IB region while trout contains the four MHC UCA, UDA, UEA and UFA class I loci. The large differences in gene content and most likely function of the salmon and trout class IB region clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa.

Expression of MHC class I pathway genes in response to infectious salmon anaemia virus in Atlantic salmon (Salmo salar L.) cells.

Infectious salmon anaemia virus (ISAV) is the causative agent of an important viral disease threatening Atlantic salmon aquaculture. Although its structure and pathogenesis is well described little is known about its immunomodulatory effects on the host. Cellular immunity is critical in the host control of virus infections, an event attributable to antigen presentation through the MHC class I pathway, whose genes are transcriptionally activated by interferons (IFN) and other cytokines. In this study we analysed the regulation and kinetics of key genes in the salmon MHC class I pathway in relation to type I IFN during ISAV infection and poly I:C stimulation in the permissive Atlantic salmon kidney cell line (ASK). As measured by quantitative real-time PCR, ISAV induced an mRNA shut-off equivalent to 2.5-5.5-fold reduced levels of housekeeping genes at 7 days post infection. Relative to this shut-off (by normalising to beta-actin) transcription increased to peak levels at 2.8-fold for MHC class I, 10-fold for beta 2 microglobulin (beta 2m), 5.9-fold for the peptide transporter ABCB2, 8.8-fold for the proteasome component PSMB8 and 4.6-fold for the proteasome component PSMB9, presumably by activation of the IFN system as a 26-fold induction was observed for type I IFN-alpha. Expression of Mx protein was also induced 17-fold at peak level. Similar kinetics and activation levels of these genes were seen in poly I:C stimulated cells. We also isolated the salmon MHC class I UBA*0301 promoter and identified a conserved interferon-stimulated response element (ISRE) and GAAA-elements plus several GAS- and IRF-sites, all supporting IFN-inducible properties. In summary, we demonstrate a concerted induction of the MHC class I pathway and type I IFN by ISAV comparable to levels induced by the synthetic double-stranded RNA (dsRNA) poly I:C. Thus, unlike influenza and several other viruses ISAV does not seem to interfere with MHC and IFN expression.

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures.

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.

Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana.

BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21,000 Arabidopsis transcripts.