High density linkage maps, genetic architecture, and genomic prediction of growth and wood properties in Pinus radiata.

BACKGROUND: The growing availability of genomic resources in radiata pine paves the way for significant advances in fundamental and applied genomic research. We constructed robust high-density linkage maps based on exome-capture genotyping in two F1 populations, and used these populations to perform quantitative trait locus (QTL) scans, genomic prediction and quantitative analyses of genetic architecture for key traits targeted by tree improvement programmes. RESULTS: Our mapping approach used probabilistic error correction of the marker data, followed by an iterative approach based on stringent parameters. This approach proved highly effective in producing high-density maps with robust marker orders and realistic map lengths (1285-4674 markers per map, with sizes ranging from c. 1643-2292 cM, and mean marker intervals of 0.7-2.1 cM). Colinearity was high between parental linkage maps, although there was evidence for a large chromosomal rearrangement (affecting ~ 90 cM) in one of the parental maps. In total, 28 QTL were detected for growth (stem diameter) and wood properties (wood density and fibre properties measured by Silviscan) in the QTL discovery population, with 1-3 QTL of small to moderate effect size detected per trait in each parental map. Four of these QTL were validated in a second, unrelated F1 population. Results from genomic prediction and analyses of genetic architecture were consistent with those from QTL scans, with wood properties generally having moderate to high genomic heritabilities and predictive abilities, as well as somewhat less complex genetic architectures, compared to growth traits. CONCLUSIONS: Despite the economic importance of radiata pine as a plantation forest tree, robust high-density linkage maps constructed from reproducible, sequence-anchored markers have not been published to date. The maps produced in this study will be a valuable resource for several applications, including the selection of marker panels for genomic prediction and anchoring a recently completed de novo whole genome assembly. We also provide the first map-based evidence for a large genomic rearrangement in radiata pine. Finally, results from our QTL scans, genomic prediction, and genetic architecture analyses are informative about the genomic basis of variation in important phenotypic traits.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.

Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

Identification of transcriptome signatures and biomarkers specific for potential developmental toxicants inhibiting human neural crest cell migration.

The in vitro test battery of the European research consortium ESNATS ('novel stem cell-based test systems') has been used to screen for potential human developmental toxicants. As part of this effort, the migration of neural crest (MINC) assay has been used to evaluate chemical effects on neural crest function. It identified some drug-like compounds in addition to known environmental toxicants. The hits included the HSP90 inhibitor geldanamycin, the chemotherapeutic arsenic trioxide, the flame-retardant PBDE-99, the pesticide triadimefon and the histone deacetylase inhibitors valproic acid and trichostatin A. Transcriptome changes triggered by these substances in human neural crest cells were recorded and analysed here to answer three questions: (1) can toxicants be individually identified based on their transcript profile; (2) how can the toxicity pattern reflected by transcript changes be compacted/dimensionality-reduced for practical regulatory use; (3) how can a reduced set of biomarkers be selected for large-scale follow-up? Transcript profiling allowed clear separation of different toxicants and the identification of toxicant types in a blinded test study. We also developed a diagrammatic system to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. activation of KEGG pathways or overrepresentation of gene ontology terms). The transcript data were mined for potential markers of toxicity, and 39 transcripts were selected to either indicate general developmental toxicity or distinguish compounds with different modes-of-action in read-across. In summary, we found inclusion of transcriptome data to largely increase the information from the MINC phenotypic test.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Analysis of central Hox protein types across bilaterian clades: on the diversification of central Hox proteins from an Antennapedia/Hox7-like protein.

Hox proteins are among the most intensively studied transcription factors and represent key factors in establishing morphological differences along the anterior-posterior axis of animals. They are generally regarded as highly conserved in function, a view predominantly based on experiments comparing a few (anterior) Hox proteins. However, the extent to which central or abdominal Hox proteins share conserved functions and sequence signatures remains largely unexplored. To shed light on the functional divergence of the central Hox proteins, we present an easy to use resource aimed at predicting the functional similarities of central Hox proteins using sequence elements known to be relevant to Hox protein functions. We provide this resource both as a stand-alone download, including all information, as well as via a simplified web-interface that facilitates an accurate and fine-tuned annotation of novel Hox sequences. The method used in the manuscript is, so far, the only published sequence-based method capable of differentiating between the functionally distinct central Hox proteins with near-identical homeodomains (such as the Drosophila Antp, Ubx and Abd-A Hox proteins). In this manuscript, a pairwise-sequence-similarity based approach (using the bioinformatics tool CLANS) is used to analyze all available central Hox protein sequences. The results are combined with a large-scale species phylogeny to depict the presence/absence of central Hox sequence-types across the bilaterian lineage. The obtained pattern of distribution of the Hox sequence-types throughout the species tree enables us to infer at which branching point a specific type of central Hox protein was present. Based on the Hox sequences currently available in public databases, seven sequence-similarity groups could be identified for the central Hox proteins, two of which have never been described before (Echi/Hemi7 and Echi/Hemi8). Our work also shows, for the first time, that Antp/Hox7-like sequences are present throughout all bilaterian clades and that all other central Hox protein groups are specific to sub-lineages in the protostome or deuterostome branches only.

PathExpress update: the enzyme neighbourhood method of associating gene-expression data with metabolic pathways.

The post-genomic era presents us with the challenge of linking the vast amount of raw data obtained with transcriptomic and proteomic techniques to relevant biological pathways. We present an update of PathExpress, a web-based tool to interpret gene-expression data and explore the metabolic network without being restricted to predefined pathways. We define the Enzyme Neighbourhood (EN) as a sub-network of linked enzymes with a limited path length to identify the most relevant sub-networks affected in gene-expression experiments. PathExpress is freely available at: http://bioinfoserver.rsbs.anu.edu.au/utils/PathExpress/.

A gene expression atlas of the model legume Medicago truncatula.

Legumes played central roles in the development of agriculture and civilization, and today account for approximately one-third of the world's primary crop production. Unfortunately, most cultivated legumes are poor model systems for genomic research. Therefore, Medicago truncatula, which has a relatively small diploid genome, has been adopted as a model species for legume genomics. To enhance its value as a model, we have generated a gene expression atlas that provides a global view of gene expression in all major organ systems of this species, with special emphasis on nodule and seed development. The atlas reveals massive differences in gene expression between organs that are accompanied by changes in the expression of key regulatory genes, such as transcription factor genes, which presumably orchestrate genetic reprogramming during development and differentiation. Interestingly, many legume-specific genes are preferentially expressed in nitrogen-fixing nodules, indicating that evolution endowed them with special roles in this unique and important organ. Comparative transcriptome analysis of Medicago versus Arabidopsis revealed significant divergence in developmental expression profiles of orthologous genes, which indicates that phylogenetic analysis alone is insufficient to predict the function of orthologs in different species. The data presented here represent an unparalleled resource for legume functional genomics, which will accelerate discoveries in legume biology.

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer.

Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species (Stigmatella, Gemmata and Methylococcus) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.

Adaptation to Host-Specific Bacterial Pathogens Drives Rapid Evolution of a Human Innate Immune Receptor.

The selective pressure by infectious agents is a major driving force in the evolution of humans and other mammals. Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as receptors for bacterial pathogens of the genera Haemophilus, Helicobacter, Neisseria, and Moraxella, which engage CEACAMs via distinct surface adhesins. While microbial attachment to epithelial CEACAMs facilitates host colonization, recognition by CEACAM3, a phagocytic receptor expressed by granulocytes, eliminates CEACAM-binding bacteria. Sequence analysis of primate CEACAM3 orthologs reveals that this innate immune receptor is one of the most rapidly evolving human proteins. In particular, the pathogen-binding extracellular domain of CEACAM3 shows a high degree of non-synonymous versus synonymous nucleotide exchanges, indicating an exceptionally strong positive selection. Using CEACAM3 domains derived from different primates, we find that the amino acid alterations found in CEACAM3 translate into characteristic binding patterns for bacterial adhesins. One such amino acid residue is F62 in human and chimp CEACAM3, which is not present in other primates and which is critical for binding the OMP P1 adhesin of Haemophilus aegyptius. Incorporation of the F62-containing motif into gorilla CEACAM3 results in a gain-of-function phenotype with regard to phagocytosis of H. aegyptius. Moreover, CEACAM3 polymorphisms found in human subpopulations widen the spectrum of recognized bacterial adhesins, suggesting an ongoing multivariate selection acting on this innate immune receptor. The species-specific detection of diverse bacterial adhesins helps to explain the exceptionally fast evolution of CEACAM3 within the primate lineage and provides an example of Red Queen dynamics in the human genome.

Bioinformatic analysis of the CLE signaling peptide family.

BACKGROUND: Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. RESULTS: We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. CONCLUSION: Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.

Analyzing microarray data using CLANS.

UNLABELLED: Analysis of microarray experiments is complicated by the huge amount of data involved. Searching for groups of co-expressed genes is akin to searching for protein families in a database as, in both cases, small subsets of genes with similar features are to be found within vast quantities of data. CLANS was originally developed to find protein families in large sets of amino acid sequences where the amount of data involved made phylogenetic approaches overly cumbersome. We present a number of improvements that greatly extend the previous version of CLANS and show its application to microarray data as well as its ability of incorporating additional information to facilitate interactive analysis. AVAILABILITY: The program is available for download from: http://bioinfoserver.rsbs.anu.edu.au/downloads/clans/

Mclip: motif detection based on cliques of gapped local profile-to-profile alignments.

UNLABELLED: A multitude of motif-finding tools have been published, which can generally be assigned to one of three classes: expectation-maximization, Gibbs-sampling or enumeration. Irrespective of this grouping, most motif detection tools only take into account similarities across ungapped sequence regions, possibly causing short motifs located peripherally and in varying distance to a 'core' motif to be missed. We present a new method, adding to the set of expectation-maximization approaches, that permits the use of gapped alignments for motif elucidation. AVAILABILITY: The program is available for download from: http://bioinfoserver.rsbs.anu.edu.au/downloads/mclip.jar. SUPPLEMENTARY INFORMATION: http://bioinfoserver.rsbs.anu.edu.au/utils/mclip/info.php.

AbrB-like transcription factors assume a swapped hairpin fold that is evolutionarily related to double-psi beta barrels.

AbrB is a key transition-state regulator of Bacillus subtilis. Based on the conservation of a betaalphabeta structural unit, we proposed a beta barrel fold for its DNA binding domain, similar to, but topologically distinct from, double-psi beta barrels. However, the NMR structure revealed a novel fold, the "looped-hinge helix." To understand this discrepancy, we undertook a bioinformatics study of AbrB and its homologs; these form a large superfamily, which includes SpoVT, PrlF, MraZ, addiction module antidotes (PemI, MazE), plasmid maintenance proteins (VagC, VapB), and archaeal PhoU homologs. MazE and MraZ form swapped-hairpin beta barrels. We therefore reexamined the fold of AbrB by NMR spectroscopy and found that it also forms a swapped-hairpin barrel. The conservation of the core betaalphabeta element supports a common evolutionary origin for swapped-hairpin and double-psi barrels, which we group into a higher-order class, the cradle-loop barrels, based on the peculiar shape of their ligand binding site.

Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation.

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.The permeabilization of the mitochondrial outer membrane to induce apoptosis is regulated by complex interactions between Bcl-2 family members. Here the authors develop a quantitative interactome of a membrane Bcl-2 network and identify a hierarchy of protein complexes in apoptosis induction.

WIPI3 and WIPI4 ß-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy.

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

Rab14 is part of the early endosomal clathrin-coated TGN microdomain.

Rab14 localizes to the Golgi/TGN and to early endosomes, but its biological function remains unclear. By structural modeling, we identified Rab14-specific residues and established a close relationship between the Rab2/Rab4/Rab14, Rab11/25 and Rab39 sub-groups within the Rab protein family. By quantitative confocal microscopy and by density centrifugation we show that Rab14 is part of the early endosomal AP-1 microdomain. Overexpression of a dominant-negative Rab14 GTP-binding mutant that solely localizes to the Golgi donor compartment accelerated EGF degradation. We suggest that the AP-1 microdomain represents the interconnecting compartment in which Rab14 vesicles cycle between early endosomes and the Golgi cisternae.

PhyloGenie: automated phylome generation and analysis.

Phylogenetic reconstruction is the method of choice to determine the homologous relationships between sequences. Difficulties in producing high-quality alignments, which are the basis of good trees, and in automating the analysis of trees have unfortunately limited the use of phylogenetic reconstruction methods to individual genes or gene families. Due to the large number of sequences involved, phylogenetic analyses of proteomes preclude manual steps and therefore require a high degree of automation in sequence selection, alignment, phylogenetic inference and analysis of the resulting set of trees. We present a set of programs that automates the steps from seed sequence to phylogeny and a utility to extract all phylogenies that match specific topological constraints from a database of trees. Two example applications that show the type of questions that can be answered by phylome analysis are provided. The generation and analysis of the Thermoplasma acidophilum phylome with regard to lateral gene transfer between Thermoplasmata and Sulfolobus, showed best BLAST hits to be far less reliable indicators of lateral transfer than the corresponding protein phylogenies. The generation and analysis of the Danio rerio phylome provided more than twice as many proteins as described previously, supporting the hypothesis of an additional round of genome duplication in the actinopterygian lineage.

Solving Classification Problems for Large Sets of Protein Sequences with the Example of Hox and ParaHox Proteins.

Phylogenetic methods are key to providing models for how a given protein family evolved. However, these methods run into difficulties when sequence divergence is either too low or too high. Here, we provide a case study of Hox and ParaHox proteins so that additional insights can be gained using a new computational approach to help solve old classification problems. For two (Gsx and Cdx) out of three ParaHox proteins the assignments differ between the currently most established view and four alternative scenarios. We use a non-phylogenetic, pairwise-sequence-similarity-based method to assess which of the previous predictions, if any, are best supported by the sequence-similarity relationships between Hox and ParaHox proteins. The overall sequence-similarities show Gsx to be most similar to Hox2-3, and Cdx to be most similar to Hox4-8. The results indicate that a purely pairwise-sequence-similarity-based approach can provide additional information not only when phylogenetic inference methods have insufficient information to provide reliable classifications (as was shown previously for central Hox proteins), but also when the sequence variation is so high that the resulting phylogenetic reconstructions are likely plagued by long-branch-attraction artifacts.