Copy number variation and population-specific immune genes in the model vertebrate zebrafish.

Copy number variation in large gene families is well characterized for plant resistance genes, but similar studies are rare in animals. The zebrafish (Danio rerio) has hundreds of NLR immune genes, making this species ideal for studying this phenomenon. By sequencing 93 zebrafish from multiple wild and laboratory populations, we identified a total of 1513 NLRs, many more than the previously known 400. Approximately half of those are present in all wild populations, but only 4% were found in 80% or more of the individual fish. Wild fish have up to two times as many NLRs per individual and up to four times as many NLRs per population than laboratory strains. In contrast to the massive variability of gene copies, nucleotide diversity in zebrafish NLR genes is very low: around half of the copies are monomorphic and the remaining ones have very few polymorphisms, likely a signature of purifying selection.

Humans and other animals have immune systems that protect them from bacteria, viruses and other potentially harmful microbes. Members of a family of genes known as the NLR family play various roles in helping to recognize and destroy these microbes. Different species have varying numbers of NLR genes, for example, humans have 22 NLRs, but fish can have hundreds. 400 have been found in the small tropical zebrafish, also known as zebra danios. Zebrafish are commonly used as model animals in research studies because they reproduce quickly and are easy to keep in fish tanks. Much of what we know about fish biology comes from studying strains of those laboratory zebrafish, including the 400 NLRs found in a specific laboratory strain. Many NLRs in zebrafish are extremely similar, suggesting that they have only evolved fairly recently through gene duplication. It remains unclear why laboratory zebrafish have so many almost identical NLRs, or if wild zebrafish also have lots of these genes. To find out more, Schäfer et al. sequenced the DNA of NLRs from almost 100 zebrafish from multiple wild and laboratory populations. The approach identified over 1,500 different NLR genes, most of which, were previously unknown. Computational modelling suggested that each wild population of zebrafish may harbour up to around 2,000 NLR genes, but laboratory strains had much fewer NLRs. The numbers of NLR genes in individual zebrafish varied greatly ­ only 4% of the genes were present in 80% or more of the fish. Many genes were only found in specific populations or single individuals. Together, these findings suggest that the NLR family has expanded in zebrafish as part of an ongoing evolutionary process that benefits the immune system of the fish. Similar trends have also been observed in the NLR genes of plants, indicating there may be an evolutionary strategy across all living things to continuously diversify large families of genes. Additionally, this work highlights the lack of diversity in the genes of laboratory animals compared with those of their wild relatives, which may impact how results from laboratory studies are used to inform conservation efforts or are interpreted in the context of human health.

Phylogenetic insight into ABCE gene subfamily in plants.

ATP-BINDING CASSETTE SUBFAMILY E MEMBER (ABCE) proteins are one of the most conserved proteins across eukaryotes and archaea. Yeast and most animals possess a single ABCE gene encoding the critical translational factor ABCE1. In several plant species, including Arabidopsis thaliana and Oryza sativa, two or more ABCE gene copies have been identified, however information related to plant ABCE gene family is still missing. In this study we retrieved ABCE gene sequences of 76 plant species from public genome databases and comprehensively analyzed them with the reference to A. thaliana ABCE2 gene (AtABCE2). Using bioinformatic approach we assessed the conservation and phylogeny of plant ABCEs. In addition, we performed haplotype analysis of AtABCE2 and its paralogue AtABCE1 using genomic sequences of 1,135 A. thaliana ecotypes. Plant ABCE proteins showed overall high sequence conservation, sharing at least 78% of amino acid sequence identity with AtABCE2. We found that over half of the selected species have two to eight ABCE genes, suggesting that in plants ABCE genes can be classified as a low-copy gene family, rather than a single-copy gene family. The phylogenetic trees of ABCE protein sequences and the corresponding coding sequences demonstrated that Brassicaceae and Poaceae families have independently undergone lineage-specific split of the ancestral ABCE gene. Other plant species have gained ABCE gene copies through more recent duplication events. We also noticed that ploidy level but not ancient whole genome duplications experienced by a species impacts ABCE gene family size. Deeper analysis of AtABCE2 and AtABCE1 from 1,135 A. thaliana ecotypes revealed four and 35 non-synonymous SNPs, respectively. The lower natural variation in AtABCE2 compared to AtABCE1 is in consistence with its crucial role for plant viability. Overall, while the sequence of the ABCE protein family is highly conserved in the plant kingdom, many plants have evolved to have more than one copy of this essential translational factor.

Sex-dependent expression levels of VAV1 and P2X7 in PBMC of multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system and the leading cause of progressive neurological disability in young adults. It decreases the patient's lifespan by about 10 years and affects women more than men. No medication entirely restricts or reverses neurological degradation. However, early diagnosis and treatment increase the possibility of a better outcome. To identify new MS biomarkers, we tested the expression of six potential markers (P2X4, P2X7, CXCR4, RGS1, RGS16 and VAV1) using qPCR in peripheral blood mononuclear cells (PBMC) of MS patients treated with interferon ß (IFNß), with glatiramer acetate (GA) or untreated. We showed that P2X7 and VAV1 are significantly induced in MS patients. In contrast, the expression of P2X4, CXCR4, RGS1 and RGS16 was not significantly modified by MS in PBMC. P2X7 and VAV1 are essentially induced in female patients, suggesting these markers are connected to sex-specific mechanisms. Strikingly, VAV1 expression is higher in healthy women than healthy men and IFNß treatment of MS reduced VAV1 expression in female MS patients while it up-regulated VAV1 in male MS patients. Our data point to the differential, sex-dependent value of MS markers and treatment effects. Although rgs16 expression in PBMC was not a valid MS marker in patients, the strong upregulation of P2X4 and P2X7 induced in the spinal cord of WT mice by EAE was abrogated in rgs16KO mice suggesting that rgs16 is required for P2X4 and P2X7 induction by neurological diseases.

Recurrent expansions of B30.2-associated immune receptor families in fish.

B30.2 domains, also known as PRY/SPRY, are key components of specific subsets of two large families of proteins involved in innate immunity: the tripartite motif proteins (TRIMs) and the Nod-like receptors (NLRs). TRIM proteins are important, often inducible factors of antiviral innate immunity, targeting multiple steps of viral cycles through a variety of mechanisms. NLRs prime and regulate systemic innate defenses, especially against bacteria, and control inflammation. Large TRIM and NLR subsets characterized by the presence of a B30.2 domain have been reported from a few fish species including zebrafish and seem to be strongly prone to gene duplication/expansion. Here, we performed a large-scale survey of these receptors across about 150 fish genomes, focusing on ray-finned fishes. We assessed the number and genomic distribution of domains and domain combinations associated with TRIMs, NLRs, and other genes containing B30.2 domains and looked for gene expansion patterns across fish groups. We then used a model to test the impact of taxonomy, genome size, and environmental variables on the copy numbers of these genes. Our findings reveal novel domain structures, clade-specific gains and losses. They also assist with the timing of the gene expansions, reveal patterns associated with the MHC, and lay the groundwork for further studies delving deeper into the forces that drive the copy number variation of immune genes on a species level.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.

The Laboratory Domestication of Zebrafish: From Diverse Populations to Inbred Substrains.

We know from human genetic studies that practically all aspects of biology are strongly influenced by the genetic background, as reflected in the advent of "personalized medicine." Yet, with few exceptions, this is not taken into account when using laboratory populations as animal model systems for research in these fields. Laboratory strains of zebrafish (Danio rerio) are widely used for research in vertebrate developmental biology, behavior, and physiology, for modeling diseases, and for testing pharmaceutic compounds in vivo. However, all of these strains are derived from artificial bottleneck events and therefore are likely to represent only a fraction of the genetic diversity present within the species. Here, we use restriction site-associated DNA sequencing to genetically characterize wild populations of zebrafish from India, Nepal, and Bangladesh, and to compare them to previously published data on four common laboratory strains. We measured nucleotide diversity, heterozygosity, and allele frequency spectra, and find that wild zebrafish are much more diverse than laboratory strains. Further, in wild zebrafish, there is a clear signal of GC-biased gene conversion that is missing in laboratory strains. We also find that zebrafish populations in Nepal and Bangladesh are most distinct from all other strains studied, making them an attractive subject for future studies of zebrafish population genetics and molecular ecology. Finally, isolates of the same strains kept in different laboratories show a pattern of ongoing differentiation into genetically distinct substrains. Together, our findings broaden the basis for future genetic, physiological, pharmaceutic, and evolutionary studies in Danio rerio.

P2X4: A fast and sensitive purinergic receptor.

Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations), about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly characterized, more studies are needed as it is likely to be a potential therapeutic target in these multiple pathologies.

The proto-MHC of placozoans, a region specialized in cellular stress and ubiquitination/proteasome pathways.

The MHC is a large genetic region controlling Ag processing and recognition by T lymphocytes in vertebrates. Approximately 40% of its genes are implicated in innate or adaptive immunity. A putative proto-MHC exists in the chordate amphioxus and in the fruit fly, indicating that a core MHC region predated the emergence of the adaptive immune system in vertebrates. In this study, we identify a putative proto-MHC with archetypal markers in the most basal branch of Metazoans--the placozoan Trichoplax adhaerens, indicating that the proto-MHC is much older than previously believed--and present in the common ancestor of bilaterians (contains vertebrates) and placozoans. Our evidence for a T. adhaerens proto-MHC was based on macrosynteny and phylogenetic analyses revealing approximately one third of the multiple marker sets within the human MHC-related paralogy groups have unique counterparts in T. adhaerens, consistent with two successive whole genome duplications during early vertebrate evolution. A genetic ontologic analysis of the proto-MHC markers in T. adhaerens was consistent with its involvement in defense, showing proteins implicated in antiviral immunity, stress response, and ubiquitination/proteasome pathway. Proteasome genes psma, psmb, and psmd are present, whereas the typical markers of adaptive immunity, such as MHC class I and II, are absent. Our results suggest that the proto-MHC was involved in intracellular intrinsic immunity and provide insight into the primordial architecture and functional landscape of this region that later in evolution became associated with numerous genes critical for adaptive immunity in vertebrates.

R4 regulators of G protein signaling (RGS) identify an ancient MHC-linked synteny group.

Regulators of G protein signaling (RGS) are key regulators of G protein signaling. RGS proteins of the R4 RGS group are composed of a mere RGS domain and are mainly involved in immune response modulation. In both human and mouse, most genes encoding the R4 RGS proteins are located in the same region of chromosome 1. We show here that the RGS1/RGS16 neighborhood constitutes a synteny group well conserved across tetrapods and closely linked to the MHC paralogon of chromosome 1. Genes located in the RGS1/RGS16 region have paralogs close to the MHC on chromosome 6 or close to the other MHC paralogons. In amphioxus, a cephalochordate, these genes possess orthologs that are located in the same scaffolds as a number of markers defining the proto-MHC in this species (Abi-Rached et al., Nat Genet 31:100-115, 2002). We therefore propose that the RGS1/RGS16 region provides useful markers to investigate the origins and the evolution of the MHC. In addition, we show that some genes of the region appear to have immune functions not only in human, but also in Xenopus.

Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed -1 ribosomal frameshifting.

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.