Cryptic MHC Polymorphism Revealed but Not Explained by Selection on the Class IIB Peptide-Binding Region

The immune genes of the major histocompatibility complex (MHC) are characterized by extraordinarily high levels of nucleotide and haplotype diversity. This variation is maintained by pathogen-mediated balancing selection that is operating on the peptide-binding region (PBR). Several recent studies have found, however, that some populations possess large clusters of alleles that are translated into virtually identical proteins. Here, we address the question of how this nucleotide polymorphism is maintained with little or no functional variation for selection to operate on. We investigate circa 750–850 bp of MHC class II DAB genes in four wild populations of the guppy Poecilia reticulata. By sequencing an extended region, we uncovered 40.9% more sequences (alleles), which would have been missed if we had amplified the exon 2 alone. We found evidence of several gene conversion events that may have homogenized sequence variation. This reduces the visible copy number variation (CNV) and can result in a systematic underestimation of the CNV in studies of the MHC and perhaps other multigene families. We then focus on a single cluster, which comprises 27 (of a total of 66) sequences. These sequences are virtually identical and show no signal of selection. We use microsatellites to reconstruct the populations' demography and employ simulations to examine whether so many similar nucleotide sequences can be maintained in the populations. Simulations show that this variation does not behave neutrally. We propose that selection operates outside the PBR, for example, on linked immune genes or on the “sheltered load” that is thought to be associated to the MHC. Future studies on the MHC would benefit from extending the amplicon size to include polymorphisms outside the exon with the PBR. This may capture otherwise cryptic haplotype variation and CNV, and it may help detect other regions in the MHC that are under selection

Iron and ferritin accumulate in separate cellular locations in Phaseolus seeds

<p>Abstract</p> <p>Background</p> <p>Iron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron.</p> <p>Results</p> <p>Here we reveal the distribution of iron in seeds of three <it>Phaseolus </it>species including thirteen genotypes of <it>P. vulgaris</it>, <it>P. coccineus</it>, and <it>P. lunatus</it>. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of <it>P. vulgaris </it>and <it>P. coccineus </it>seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 μg g^-1of iron, depending on the genotype. In contrast to <it>P. vulgaris </it>and <it>P. coccineus</it>, we did not observe iron accumulation in the cells surrounding the provascular tissues of <it>P. lunatus </it>cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype.</p> <p>Conclusions</p> <p>The presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.</p

A Set of Lotus japonicus Gifu × Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype ‘Gifu’ was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation

t2prhd: a tool to study the patterns of repeat evolution

BACKGROUND: The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading. RESULTS: We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences. CONCLUSION: Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of script

Brain and Pituitary Response to Vaccination in Gilthead Seabream (Sparus aurata L.)

Vaccination is a widely used therapeutical strategy in aquaculture, but whether vaccination elicits stress responses in the central neuroendocrine system and enhances the crosstalk between the immune and endocrine systems in the brain or pituitary after vaccination is unclear. To answer this question two experiments using two different vaccine exposure routes, i.e., bath or intraperitoneal (i.p.) injection, were carried out on gilthead seabream (Sparus aurata L.). In the first one, the stress responses of fish subjected to waterborne Vibrio anguillarum bacterin were compared with responses after air exposure or their combination. In the second experiment, fish were subjected to an intraperitoneal injection of Lactococcus garvieae bacterin and we assessed the central stress response and also whether or not a significant immune response was induced in brain and pituitary. In both experiments, blood, brain and pituitary tissues were collected at 1, 6, and 24 h post stress for plasma hormone determination and gene expression analysis, respectively. Results indicated that bath vaccination induced a decreased central stress response compared to air exposure which stimulated both brain and pituitary stress genes. In the second experiment, injection vaccination kept unchanged plasma stress hormones except cortisol that raised at 6 and 24 h. In agreement, non-significant or slight changes on the transcription of stress-related genes were recorded, including the hormone genes of the hypothalamic pituitary interrenal (HPI) axis and other stress markers such as hsp70, hsp90, and mt genes in either brain or pituitary. Significant changes were observed, however, in crhbp and gr. In this second experiment the immune genes il1b, cox2, and lys, showed a strong expression in both brain and pituitary after vaccination, notably il1b which showed more than 10 fold raise. Overall, vaccination procedures, although showing a cortisol response, did not induce other major stress response in brain or pituitary, regardless the administration route. Other than main changes, the alteration of crhbp and gr suggests that these genes could play a relevant role in the feedback regulation of HPI axis after vaccination. In addition, from the results obtained in this work, it is also demonstrated that the immune system maintains a high activity in both brain and pituitary after vaccine injection

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Dosage compensation in Drosophila melanogaster involves the selective targeting of the male X chromosome by the dosage compensation complex (DCC) and the coordinate, ∼2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize ‘chromosomal entry sites’ or ‘high-affinity sites’ may serve such a function. However, to date no DNA binding domain that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2–MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo

Recessive Antimorphic Alleles Overcome Functionally Redundant Loci to Reveal TSO1 Function in Arabidopsis Flowers and Meristems

Arabidopsis TSO1 encodes a protein with conserved CXC domains known to bind DNA and is homologous to animal proteins that function in chromatin complexes. tso1 mutants fall into two classes due to their distinct phenotypes. Class I, represented by two different missense mutations in the CXC domain, leads to failure in floral organ development, sterility, and fasciated inflorescence meristems. Class II, represented by a nonsense mutation and a T-DNA insertion line, develops wild-type–like flowers and inflorescences but shows severely reduced fertility. The phenotypic variability of tso1 alleles presents challenges in determining the true function of TSO1. In this study, we use artificial microRNA, double mutant analysis, and bimolecular fluorescence complementation assay to investigate the molecular basis underlying these two distinct classes of phenotypes. We show that the class I mutants could be converted into class II by artificial microRNA knockdown of the tso1 mutant transcript, suggesting that class I alleles produce antimorphic mutant proteins that interfere with functionally redundant loci. We identified one such redundant factor coded by the closely related TSO1 homolog SOL2. We show that the class I phenotype can be mimicked by knocking out both TSO1 and its homolog SOL2 in double mutants. Such antimorphic alleles targeting redundant factors are likely prevalent in Arabidopsis and maybe common in organisms with many sets of paralogous genes such as human. Our data challenge the conventional view that recessive alleles are always hypomorphic or null and that antimorphic alleles are always dominant. This study shows that recessive alleles can also be antimorphic and can produce a phenotype more severe than null by interfering with the function of related loci. This finding adds a new paradigm to classical genetic concepts, with important implications for future genetic studies both in basic research as well as in agriculture and medicine

Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

<p>Abstract</p> <p>Background</p> <p><it>Phytophthora infestans </it>is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The <it>P. infestans </it>genome experienced a repeat-driven expansion relative to the genomes of <it>Phytophthora sojae </it>and <it>Phytophthora ramorum </it>and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation.</p> <p>Results</p> <p>We used <it>in silico </it>approaches to predict and describe the repertoire of <it>P. infestans </it>secreted proteins (the secretome). We defined the "plastic secretome" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the <it>P. sojae </it>and <it>P. ramorum </it>genomes and (iii) is encoded by genes residing in gene sparse regions of <it>P. infestans </it>genome. Although including only ~3% <it>of P. infestans </it>genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced <it>in planta</it>. We highlight 19 plastic secretome genes induced <it>in planta </it>but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors.</p> <p>Conclusions</p> <p>This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes.</p