Pyrococcus furiosus (Pfu). The 2.5 Å resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely α-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 Å resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19•SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon  In all living cells the signal recognition particle (SRP) recognizes nascent polypeptides destined for secretion or membrane insertion as they emerge from translating ribosomes In eukaryotes, the SRP contains six proteins  and a 300-nucleotide RNA. Most bacterial systems display a simplest organization with a shorter RNA (about 113 nucleotides) and a single protein subunit Ffh, the homologue of SRP54. Archeal SRPs contain an SRP RNA of similar size and fold to that in eukaryotes but only two proteins, SRP54 and SRP19 (ras-like fold) and the C-terminal M domain (for methionine rich). While the N and G domains associate together to constitute a structural and functional catalytic core, the M domain is responsible for the promiscuous recognition of the diverse signal sequences and binding to the helix 8 of the SRP RNA. A flexible linker relates the catalytic NG core to the signal peptide and RNA-binding M domain , a central GTPase domain  to a conserved region corresponding to the so-called core RNA  present in all SRP54 sequences and located between the helix α7 at the C-terminal part of the G domain and the linker. These conserved glycine residues act as pivot points allowing the M domain to sample diverse conformation while it scans for signal sequences as they emerge from the ribosomal exit tunnel. The affinity of SRP for presecretory proteins has been shown to be dependent on nascent chain length In the two structures of Thermus, Sulfolobus, Methanococcus and now Pyrococcus represent a sampling of the conformations adopted by SRP-GTPase. Biochemical solution studies using fluorescence resonance energy transfer E.coli undergoes major conformational rearrangements upon interaction with its SRP RNA and its cognate receptor. In the tetragonal crystals of Pfu-SRP54 containing two monomers for a total solvent content of 69%, we cannot rule out the possibility that the configuration of the M domain relatively to the NG core is at least partially affected by crystalline packing interactions. This observation can indeed be made for all other crystal structures of free SRP54 reported so far Pfu and Ssol SRP54s are quite similar (high concentration of lithium sulfate as precipitating agent in acetate buffer at an acidic pH but no detergent in our case). Although as we mentioned earlier and in contrast with the SRP54 structure from Ssol there are no contacts established between the NG and the M domains within a monomer of Pfu-SRP54, analysis of the crystallographic contacts shows interactions between the NG domain and the M domain of a crystallographically symmetry-related molecule  that was not observed in the electron density maps. The domain is organized around helices α10 through α14. A groove flanked by helices α10, α11 and α14 and the finger loop constitutes the putative signal sequence binding site 2, considerably higher than the 55 Å2 observed for the NG domains; this intrinsic flexibility has been proposed to be important for the ability to recognize diverse signal sequences at different states of exit from the ribosome.The M domain of E.coliMja M domain structures bound to the core of the SRP RNA TaqSsolSsol, the finger loop appears to be defined and collapsed inside the hydrophobic groove, thus its M domain adopts a “closed” state where the finger loop folds back in the signal-binding grove shielding it from the solvent. In Taq the hydrophobic groove of the M domain is not empty but instead occupied by the finger loop from a neighboring molecule and might also contain some detergent from the crystallization solution. This is referred as an “open” state, primed for signal sequence binding. To date several structures of M domains have been reported but none has been solved in presence of a bona-fide signal sequence although a human SRP54 M domain structure Disorder in the finger loop was also observed in the Pfu-SRP54 M domain conformation is similar to the “open” state  diminished the rate of stimulated GTPase activity of the SRP•FtsY complex. In the M domain finger loop region, mutations L350D and L354D (corresponding to the non-observed finger loop positions I349 and I353 in Pfu) impaired SRP RNA-catalyzed Ffh•FtsY complex formation without notably affecting RNA binding. This region is thus likely to sense the occupancy state of the binding groove and induce structural changes to the rest of the structure, probably mediated through the SRP RNA and the GM linker.Superposition of all M domain structures available in presence or absence of SRP RNA show that the free Pfu-SRP54 was co-crystallized in presence of GDP, and the nucleotide is clearly identified in the binding cleft located in the G domain ( and Pfu-SRP54 is the first full-length SRP54 structure bound to GDP. Taq is the only other SRP54 GTPase whose GDP-bound structure is known Taq-FfhNG•Mg•GDP structure Pfu) shifted away and establishing an ionic interaction with the strictly conserved Arg191 in motif II (Arg193 in Pfu). In Pfu however, residue Asp137 from motif II is brought closer (about 6.3 Å) to the nucleotide in a conformation and geometry similar to the one described in a more recent Taq-FfhNG•Mg•GDP structure The paired hydrogen bonds between the carboxylate group of Asp250 in motif IV and the N1 and N2- nitrogens of the guanine ring are canonical nucleotide-specificity determinant seen in all SRP-GTPases Pfu structure, conserved residues Arg288 and Arg292 of the α7 anchoring-helix are well defined and solvent exposed ( and Ssol and Taq). In the Mja-SRP complex Taq-FtsY•FfhNG complex Pfu) and the DARGG motif links its position with the positioning of the conserved residue Asp248 (Asp250 in Pfu) that is directly involved in nucleotide recognition Pfu) and Glu280 (Glu282 in Pfu). From analysis of the GDP-bound Pfu-SRP54 structure we can also hypothesize that rearrangement of a cluster of hydrophobic residues contributed by helices α6 (Leu259), α7 (Phe289 and Leu293) and the β-strands β7 (Ile272) and β8 (Phe284) will also affect the geometry within the catalytic site in response to structural changes occurring in the M domain and/or the SRP RNA. Repositioning of this basic ladder from the solvent exposed conformations, observed in the free SRP54 or entire SRP, to the buried conformation observed in the FtsY•FfhNG complex  by SRP to the acquisition of the proper NG-M configurations required for the targeting process. It probably acts as a “sensor spring” and a lever able to transfer the conformational changes between the M and SRP RNA partners and the GTP catalytic center as they respectively interact with their cognate ligands .Pfu-SRP19 and determined their structures  in contact with the SRP RNA. As L2 folds, it not only establishes a conserved subset of interactions with the RNA but also some weak interactions with the M domain of SRP54/Ffh as observed in the more “complete” complex structures from Mja that also includes SRP54  and Mja-SRP RNA The structures of SRP19 bound to SRP RNA are known in two organisms: The human SRP19 has been crystallized in presence of a short RNA fragment corresponding to the helix6 of the SRP RNA Time resolved foot-printing and fluorescence resonance energy transfer performed on the assembly of the human SRP revealed the existence of obligatory intermediates during the binding of SRP19 to SRP RNA Pfu-SRP19 supports the idea that this subunit acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54 and thus ensuring the proper maturation and assembly of a functional SRP competent for protein-targeting through the interaction with its cognate receptor. Such mutual accommodation between protein and RNA-binding surfaces is a common theme in protein-nucleic acid interactions Our present work on the free Pyrococcus furiosus. Combined with our structure of Pfu-SR (submitted), this is to our knowledge the first case where all structures of the proteins constituting the SRP-dependent protein-targeting machinery from the same archeal organism are available. Pfu-SRP54 appears as a flexible molecule with a stable though dynamic M domain articulated on a flexible linker that connects it to the NG catalytic core. The linker region of SRP54 can adopt a variety of conformations that enable the signal peptide-binding domain to scan for diverse signal sequences as they emerge from the ribosomal protein synthesis exit tunnel and also regulates the activity of the GTPase core domain by coupling these two physically separated yet functionally interconnected domains. The structure of free Pfu-SRP19 reveals a compact entity and suggests that this subunit acts as a molecular scaffold and a chaperone involved in assembly of the functional ribonucleoprotein particle. Through an induced fit mechanism involving the rearrangement of its disordered L2 loop, SRP19 assists proper folding of the SRP RNA and therefore favors subsequent binding of SRP54.We describe the X-ray structures of SRP54 and SRP19 the two protein constituting the proteinaceous core of the SRP from the hyperthermophilic archaeon Pyrococcus furiosus as a model system provides a platform suitable for further structural investigations of higher order complexes that can offer new insights into the mechanisms of the eukaryotic protein-targeting machinery.SRP54 and SRP19 constitute the conserved core in all archaeal and eukaryotic SRPs. When compared with the somewhat simpler bacterial homologues, archaeal SRPs represent an increased level of complexity in terms of structural organization and mechanism of action. However, the intrinsic robustness and stability of their constituents in the case of thermophilic organisms may constitute an advantage. The archaeon Pfu-SRP54 (PF1731) and Pfu-SRP19 (PF1894) from Pyrococcus furiosus were amplified by PCR from total genomic DNA. The Pfu-SRP54 and Pfu-SRP19 genes were respectively cloned into the pET28b and pET29b vectors (Novagen) and the corresponding proteins expressed as fusions with either N- or C- terminal hexahistidine tags cleavable with thrombin. Native proteins were expressed in BL21(DE3)-rosetta2 E.coli cells grown in auto-induction media E.coli cells grown in minimal media with glucose as carbon source and using the amino acid pathway starvation method Genes encoding the full-length proteins −1 for Pfu-SRP54 and 15 mg.ml−1 for Pfu-SRP19. Crystals of Pfu-SRP54 and Pfu-SRP19 were obtained at room temperature from a variety of conditions in hanging drops by the vapor diffusion method using a Mosquito nanoliter-scale robotic workstation (TTP Labtech). Crystals of Pfu-SRP54  grew in 1.0–1.3 M lithium sulfate and 100 mM sodium acetate pH = 5.0 with two monomers per asymmetric unit and a solvent content of 69%. Two crystal forms of Pfu-SRP19 were obtained in the same condition depending on whether the protein was native or seleno-labeled. Crystals of Pfu-SRP19 (monoclinic P21 or orthorhombic C2221 space groups) grew in 1.2–1.3 M sodium malonate and 100 mM sodium acetate pH = 5.0. The monoclinic asymmetric unit contained four monomers for a solvent content of 42% whereas the orthorhombic asymmetric unit contained two monomers for a solvent content of 34%.For crystallization, proteins were concentrated to 20 mg.mlPfu-SRP54 was solved using the anomalous dispersion signal of selenium at its excitation wavelength. The structure of the Pfu-SRP19 was solved using the anomalous dispersion of bromine at its excitation wavelength. The tetragonal crystals of Pfu-SRP54 cryo-protected in ethylene glycol diffracted to 2.5 Å. Both the monoclinic and orthorhombic crystal forms of Pfu-SRP19 were cryo-protected in ethylene glycol and diffracted to at least 1.9 Å resolution. Data were indexed, reduced and scaled with HKL2000MOSFLMScalaElvesPhenixPhaserCootX-ray diffraction data were collected at beamline 8.3.1 at the Advanced Light Source  on Quantum 210 or 315r CCD detectors. The structure of Pfu-SRP54 structure was determined by MR/Se-SAD at 3.3 Å resolution and refined to 2.5 Å. The phasing process was improved through the combination of anomalous Patterson and Fourier differences, based on partial molecular replacement solutions obtained using various NG domain structures  as search models to locate seleniums and improve phases. These molecular replacement solutions were used to determine and verify the positions of the selenium sites in the NG domain based on sequence alignments. We did not use any of the available M domain structures. Initial phases were determined 3.3 Å after density modification, non-crystallographic symmetry averaging and solvent flattening in Phenix. Ten of the twelve expected selenium sites per monomer could be located unambiguously. Following initial location of the selenium scatterers, the figure of merit was 0.35 and was further improved to 0.67 after density modification. Inspection of the initial density map at 3.3 Å resolution allowed us to trace the linker between the NG and M domains without any ambiguity and revealed the presence of contaminating GDP bound to the protein at a low occupancy. The final 2.5 Å resolution native data set was collected on a crystal grown in presence of 10 mM GDP. Initial refinement cycles included non-crystallographic symmetry restraints that were removed at the final stage. The disordered finger-loop region encompassing residues Leu346 through Ile365 is missing in the final model.The Pfu-SR19 proved inconsistent and attempts to grow crystals were particularly frustrating. Crystals of seleno-labeled protein were eventually grown in the same condition obtained for the native protein however they belong to the orthorhombic space group C2221 and despite the collection of a Se-SAD data set on a single crystal, we were unable to solve this structure using the observable anomalous signal. The Pfu-SRP19 structure (monoclinic form) was solved using a Br-SAD data set collected on a single native crystal briefly soaked in mother liquor supplemented with cryo-protectant and sodium bromide Phenix with refined occupancies ranging from 0.62 to 0.26. After initial location of the bromide scatterers, the figure of merit was 0.46 and was further improved to 0.67 after density modification. We eventually solved and refined the orthorhombic crystal form of Pfu-SRP19 by molecular replacement using the structure obtained with the monoclinic crystal form. Both structures were refined without non-crystallographic symmetry restraints. The disordered loop L2 region encompassing residues Asn57 through Glu66 is missing in the final models. Structure quality was assessed using MolProbityThe expression of seleno-substituted Figure S1Experimental phasing of SRP54. Stereo view of the initial unbiased likelihood-weighted electron density map after MR-SAD phasing and density modification at 3.3 Å resolution contoured at 1.5σ showing the linker region between the NG and M domains. The backbone trace of the final model, refined against the 2.5 Å resolution native data set, is shown placed in density. The aN1 helix of the N domain, the α7 C-terminal helix of the G domain and the G to M linker region of helix α8 are labeled and colored in green, pink and yellow, respectively. The backbone positions of residues Arg288, Arg292, Gly295 and Gly297 are indicated. Arginines Arg288 and Arg292 constitute the so-called “basic ladder” and Glycines Gly295 and Gly297 are the pivot residues involved in the relative positioning of the M and NG domains. For the sake of clarity the trace of a symmetry-related molecule is not represented.(2.49 MB TIF)Click here for additional data file.Figure S2Analysis of crystallographic contacts between the NG and the M domains. (A) Overall arrangement showing the NG domain of one monomer  and the GM-linker and M domain of its symmetry-related molecule (in blue). In this relative configuration, the distance between the end of the NG domain (Leu296 at the C-terminus of helix α7) and the N-terminus of the M domain (Gly326 at the C-terminus of helix α9) is 26 Å; the two boxed areas correspond to the only contact surfaces between the G and M domain (upper box) and the N and the G-M linker (lower box). (B) and (C) Close- up views of the two main contact areas. Residues involved in hydrogen bonding, van der Waals or ionic interactions are labeled. a-helices have been numbered.(2.94 MB TIF)Click here for additional data file.


The birds-of-paradise (Paradisaeidae) form one of the most prominent avian examples of sexual selection and show a complex biogeographical distribution. The family has accordingly been used as a case-study in several significant evolutionary and biogeographical syntheses. As a robust phylogeny of the birds-of-paradise has been lacking, these hypotheses have been tentative and difficult to assess. Here we present a well supported species phylogeny with divergence time estimates of the birds-of-paradise. We use this to assess if the rates of the evolution of sexually selected traits and speciation have been excessively high within the birds-of-paradise, as well as to re-interpret biogeographical patterns in the group.The phylogenetic results confirm some traditionally recognized relationships but also suggest novel ones. Furthermore, we find that species pairs are geographically more closely linked than previously assumed. The divergence time estimates suggest that speciation within the birds-of-paradise mainly took place during the Miocene and the Pliocene, and that several polygynous and morphologically homogeneous genera are several million years old. Diversification rates further suggest that the speciation rate within birds-of-paradise is comparable to that of the enitre core Corvoidea.The estimated ages of morphologically homogeneous and polygynous genera within the birds-of-paradise suggest that there is no need to postulate a particularly rapid evolution of sexually selected morphological traits. The calculated divergence rates further suggest that the speciation rate in birds-of-paradise has not been excessively high. Thus the idea that sexual selection could generate high speciation rates and rapid changes in sexual ornamentations is not supported by our birds-of-paradise data. Potentially, hybridization and long generation times in polygynous male birds-of-paradise have constrained morphological diversification and speciation, but external ecological factors on New Guinea may also have allowed the birds-of-paradise to develop and maintain magnificent male plumages. We further propose that the restricted but geographically complex distributions of birds-of-paradise species may be a consequence of the promiscuous breeding system. Birds-of-paradise (Paradisaeidae) are renowned for their complex courtships and diverse male plumages with highly elongated and elaborate feathers. Arguably, the birds-of-paradise form one of the most extravagant clades of all bird families in terms of beauty and display behaviour. The degree of sexual dimorphism found in the majority of species of birds-of-paradise  is one of the most prominent avian examples of sexual selection. The birds-of-paradise also show complex biogeographical patterns, where most species and subspecies are locally and disjunctly distributed, sometimes in seemingly uniform and interconnected landscapes (summarized in ). ConseqMacgregoria pulchra) is a giant honeyeater [Melampitta lugubris) and the silktail (Lamprolia victoriae) have also been proposed to be related to the birds-of-paradise [Several incongruent phylogenetic hypotheses have been proposed for the birds-of-paradise based on both morphology -8 and moneyeater , while tneyeater -15. The paradise ,16,17, bparadise ,15,18.The Fisherian runaway model of sexual selection ,20 suggestricto sensu), based on both nuclear and mitochondrial sequence data. We use this i) to asses if the speciation rate and the evolution of sexually selected traits have been excessively high in the polygynous core birds-of-paradise, and ii) to examine biogeographical patterns and analyze the geographical differentiation in relation to sexually selected traits.Here we present a robust phylogenetic hypothesis and divergence time estimates for the birds-of-paradise (b gene and the two nuclear loci ornithine decarboxylase introns 6 to 7 (ODC), and glyceraldehyde-3-phosphodehydrogenase intron 11 (GAPDH). The taxon sampling includes all 40 species of birds-of-paradise recognized by Monroe and Sibley [Loboparadisea, Cnemophilus, Melampitta and Macgregoria, which have been shown to not be part of the birds-of-paradise clade [Corvus cornix and Monarcha melanopsis, two taxa representing lineages that have been suggested to be closely related to the birds-of-paradise [We examined the phylogenetic relationship among birds-of-paradise by analyzing DNA sequence data from the mitochondrial cytochrome d Sibley , omittinse clade -15. The paradise . Three aet al. [For extractions, amplifications, and sequencing procedures from study skin samples we followed the procedures described in Irestedt et al. . SeveralWe used Bayesian inference  to estib gene, a concatenated data set of the nuclear loci (GAPDH and ODC), and a concatenated data set of all genes. In the analyses of the concatenated data sets the models selected for the individual gene partitions were used. We used an unconstrained, exponential branch length prior. All chains were run for 25 million generations, with trees sampled every 100 generations. The trees sampled during the burn-in  were discarded, and after checking for convergence, final inference was made from the concatenated output from the two runs.Posterior probabilities of trees and parameters in the substitution models were approximated with MCMC and Metropolis coupling using the program MrBayes 3.1.1 . AnalyseCorvus node  and multiply this age with our age estimate of the corresponding split. We consequently used the method to calculate diversification rates for crown groups [Sexual selection has been considered a driving force behind speciation -25. By cn groups .Acanthisitta-lineage from all other passerines [We used a relaxed clock model implemented in Beast 1.4.7 -44 to essserines ,46. The sserines ,48, but sserines ,50. In ob gene was sequenced. As we used partially overlapping primers the region covers a 868 bp region (ending 125 bp from the 3' end in the cytochrome b gene). Taking into account that for some taxa a few short fragments are missing, the alignments of the non-coding intron regions are 387 bp for GAPDH , and 685 bp for ODC . Some indels in more variable regions were found to be autapomorphic, while most other indels are congruent with the phylogenetic tree obtained from the analysis of the combined data set. The only exceptions are a 4 bp deletion shared between all Paradisaea and Monarcha and a 1 bp insertion shared by the Parotia-Pteridophora clade and some outgroup taxa in the GAPDH locus.For all taxa 841 bp of the cytochrome b, the HKY+ Γ for GAPDH, and GTR+Γ for ODC. After discarding the burn-in phase the final inference was based on a total of 200 000-225 000 samples from the posterior for all conducted Bayesian analyses. For the phylogenetic inference of the concatenated data set of all genes, the mode of the posterior distribution of topologies is presented as a 50% majority-rule consensus tree . Most deviant in relation to the combined tree is the sister relationship between Epimachus and Drepanornis (pp = 0.83) suggested only by the cytochrome b tree. In the cytochrome b tree Monarcha is also nested between the Manucodia, Phonygammus and Lycocorax clade and core birds-of-paradise (pp = 0.90).The trees obtained from the analyses of cytochrome Most nodes in our combined phylogeny receive strong support (pp > 0.95) and are often recovered by both the nuclear and the mitochondrial genes. Thus, we consider our combined phylogeny a reliable estimate of the evolutionary relationships among the birds-of-paradise.Manucodia and Phonygammus) and the paradise crow (Lycocorax pyrrhopterus). This clade is supported as the sister group to the core birds-of-paradise (remaining genera). The king of Saxony bird-of-paradise  and parotias (Parotia) form the second clade (B). Within this clade P. carolae is highly divergent and warrants separate generic treatment.Five main clades are recognized in our phylogeny , Drepanornis sicklebills, the standardwing bird-of-paradise , riflebirds (Ptiloris), and the superb bird-of-paradise (Lophorina superba). However, support for more basal nodes within this clade is low, and the affinity of the twelve-wired bird-of-paradise, Drepanornis sicklebills, and the standardwing bird-of-paradise should be regarded as provisional. Although support values are low, New Guinean Ptiloris and Lophorina form a clade separate from the Australian Ptiloris. This apparent relationship is worth investigating further as it suggests a significant phylogenetic break between Australia and New Guinea.The third clade (C) consists of the twelve-wired bird-of-paradise (Epimachus), paradigallas  and astrapias (Astrapia). The fifth clade (E), consists of Paradisaea birds-of-paradise, Wilson's bird-of-paradise (Diphyllodes respublica), magnificent bird-of-paradise (Diphyllodes magnificus), and king bird-of-paradise (Cicinnurus regius). Within this clade the genera Diphyllodes and Cicinnurus are sister lineages, while the blue bird-of-paradise (Paradisaea rudolphi) is sister to other Paradisaea birds-of-paradise.The fourth clade (D) includes sicklebills . Similarly, the morphological variation is modest within the genus Parotia, for which the exploded lekking system is estimated to be around 10 million years old. Other sexually dimorphic and polygynous genera such as Ptiloris and Astrapia also show little morphological diversification between species. The calculated diversification rates , and modest speciation and phenotypic differentiation rates are thus difficult to explain by hybridization. Although little is known about the age when birds-of-paradise breed for the fist time, it is assumed that males of polygynous species do not reproduce until they have obtained full adult plumages, which happens several years later than in females [Among passerine birds there is probably no other family with so many reported cases of interspecific and intergeneric hybrids in the wild as in the birds-of-paradise . For som females . A longeParadigalla.Birds-of-paradise are among the few bird groups that have developed a social system based on promiscuous mating and arenas or leks . It is tLycocorax pyrrhopterus and Semioptera wallacii) inhabiting Halmahera and surrounding islands of the North Moluccas. The present distributions can largely be explained by vicariance, considering the fluctuating sea levels in the Late Miocene and Pliocene [Groups closely related to the birds-of-paradise, such as Monarchidae, Rhipiduridae and Dicruridae ,55-57 haPliocene . This raManucodia, Phonygammus, Paradigalla and Lycocorax. These species are monogamous and form pair bonds, which is quite unlike the promiscuous behaviour of the rest of the family. This less "spectacular" life strategy probably enabled these birds to have a higher dispersal capacity and could explain the present day occurrence of Manucodia and Phonygammus on several islands off the New Guinea coast and on Australia's Cape York Peninsula, and of Lycocorax on the North Moluccas. Paradigalla on the other hand is a genus restricted to the central New Guinean mountain range, which renders it less likely to disperse. The present day distribution of Semioptera wallacii in the North Moluccas is more difficult to explain. However, molecular clock estimates  is evident in several other families with elaborate plumages, such as Phasianidae , PipridaAstrapia, Ptiloris and Paradigalla) there are distinct allopatric clades distributed in the east and the west of New Guinea, which separated about 3 - 6 million years ago  are slightly older. However, the divergences involving the mountain species (P. guilielmi and the highly divergent P. rudolphi) are much older. Within Ptiloris, the time separation of New Guinean and Australian taxa corresponds to broad land connections in the upper Miocene and marine transgression in the Pliocene [Within several lineages of birds-of-paradise  for which assumed sister relationships suggest a strong biogeographical connection between the Vogelkop and the Huon Peninsula in western and eastern New Guinea, respectively. Our phylogeny does not provide support for this biogeographical scenario. A sister relationship between Parotia sefilata in the Vogelkop and Parotia wahnesi on the Huon Peninsula is only weakly supported (PP = 0.59), and within the genus Astrapia our phylogeny strongly suggests sister relationships between species that occur in closely connected geographical areas . Within the genus Paradisaea, P. guiliemi from the Huon Peninsula is sister to all other Paradisaea species  occupying almost all lowland areas in New Guinea.Heads -65 has aDrepanornis as well as the two species of Epimachus separated about 10 and 7 million years ago, respectively. While the two species of Drepanornis occupy different elevations in low- and mid-montane forests, the two species of Epimachus are altitudinal replacements in mountain forests. Consequently, these two cases could represent old cases of altitudinal speciation. Parotia lawesii and Parotia helenae have similar patchy distributions as Epimachus across the mountains of New Guinea, and may represent a recent altitudinal speciation event. Examples of presumed altitudinal speciation in New Guinea have also been reported in Meliphagidae honeyeaters [The two species of eyeaters .The phylogenetic results suggest that the birds-of-paradise could be subdivided into five main clades (A-E in Figure MI designed the study, carried out the labwork, performed the phylogenetic analyses, and drafted the manuscript. PE and KJ assisted with the phylogenetic analyses and conceived the study. JF, KJ and LC assisted with the design of the study and with the draft of the manuscript. All authors read and approved the manuscript.


The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD. x structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 Å. With GDP and AlFx, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlFx requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF MnmE is an evolutionary conserved G protein that is involved in modification of the wobble U position of certain tRNAs to suppress translational wobbling. Despite high homology between its G domain and the small G protein Ras, MnmE displays entirely different regulatory properties to that of many molecular switch-type G proteins of the Ras superfamily, as its GTPase is activated by nucleotide-dependent homodimerization across the nucleotide-binding site. Here we explore the unusual G domain cycle of the MnmE protein by combining X-ray crystallography with pulse electron paramagnetic resonance (EPR) spectroscopy, which enables distance determinations between spin markers introduced at specific sites within the G domain. We determined the structures of the full-length MnmE dimer in the diphosphate and triphosphate states, which represent distinct steps of the G domain cycle, and demonstrate that the G domain cycle of MnmE comprises large conformational changes and domain movements of up to 18 Å, in which the G domains of the dimeric protein traverse from a GDP-bound open state through an open/closed equilibrium in the triphosphate state to a closed conformation in the transition state, so as to assemble the catalytic machinery. Arg(UCU), tRNAGln(UUG), tRNAGlu(UUC), tRNALeu(UAA), and tRNALys(UUU) 5U34) together with a thiolation at the 2 position favours the interaction with A and G, but suppresses base-pairing with C and U Cells devote substantial biosynthetic effort and resources to posttranscriptional modification of tRNAs Thermotoga maritima reveals a three-domain protein consisting of an N-terminal tetrahydrofolate-binding domain, a central helical domain, and a canonical Ras-like G domain inserted into the helical domain The crystal structure of MnmE from +-stimulated GTP hydrolysis x)  protein to drive the nucleotide exchange and a GTPase activating protein (GAP) to stimulate hydrolysis So far, neither the structural model of the full-length MnmE dimer nor dimerization of the G domains in the context of the full-length dimer have been proven directly. With the architecture of the proposed dimer model, dimerization of the G domains would require large domain movements suggesting that large conformational rearrangements of the protein are coupled to its GTPase cycle x and guanosine-5′-triphosphate (GppCp), and K+ and were found to crystallize readily in diverse conditions, but only in three cases—Chlorobium tepidum MnmE (CtMnmE) in the presence of K+, GDP, or GDP-AlFx; Nostoc MnmE (NoMnmE) in the presence of K+, GDP, or GDP-AlFx; and CtMnmE in the presence of K+ and GppCp-crystals with sufficient diffraction quality were obtained. In the case of CtMnmE, a polyethylene glycol (PEG) 6000/NaCl-condition produced diffraction quality crystals in the presence of GPD and GDP-AlFx. Crystals had the same unit cell parameters and the same space group and are thus isomorphous. NoMnmE crystals with sufficient diffraction were obtained in a PEG 550 monomethyl ether (MME) condition. As with CtMnmE, crystals obtained in the presence of GDP-AlFx or GDP were isomorphous. Structure determination showed in both cases that the crystals contained the GDP-bound form of MnmE, despite the presence of AlFx. Quality of crystals grown in the presence of GDP-AlFx were somewhat better, hence their datasets were used for structure determination.Various MnmE homologous have been screened for crystallization conditions in the presence of GDP, GDP-AlFx) crystallized in the space groups I4(1)22 and P4(3)2(1)2, respectively, each with one full length protomer in the asymmetric unit. In both cases homodimers are formed via crystallographic symmetry by means of the N-terminal domains . Molecules B and C form a dimer within the asymmetric unit, while protomer A forms a dimer with its crystallographic symmetry mate shown in . No densx and GDP were used in the crystallization trials, the G domains were separated from each other by large distances. They do not display any structural contacts between each other nor to the N-terminal or helical domains. In all the structures, nucleotides are far apart from each other, with distances of 38 to 56 Å between the first P-loop glycines' Cα atom (GxxxxGKS motif).The overall homodimer architecture found in the three structures resembles the proposed model obtained from a partial dimer , with th2+ is coordinated to the phosphates, and switch I-contacts to GDP are absent , the other occupies the usual Mg2+-binding site at the β-phosphate of GDP  or switch II (NoMnmE·GDP) disordered and thus not resolved. Nucleotides are bound in a way typical for Ras-like G domains –S1E. In e absent . In NoMne of GDP . As Switx-in the γ-phosphate binding site mimics the transition state of the phosphor transfer reaction and is considered the litmus test for correct assembly of the active site. In the case of MnmE, this is thought to be achieved by dimerization and close juxtaposition of the two G domains across the nucleotide binding site, as observed for the isolated G domains x in the crystallization trials, no electron density for AlFx could be observed -GDP (mGDP) bound to MnmE upon addition of AlFT. maritima MnmE dimer model, generated with pdb 1XZP) reveals that the N-terminal domains align quite well and only minor displacements are present for the helical domains  into the asymmetric unit of the CtMnmE·GppCp structure to the position expected for the G domain of molecule C would create a much smaller hypothetical crystal contact area of only 18 Å2 (or 131 Å2). Thus we would expect that the G domain of molecule C is present in the crystal but, due to its high mobility and absence of sufficient crystal contacts, is not visible in the electron density map. This is similar to the recent structure of the Roco protein, which is also a GAD protein. There, the second G domain of the constitutive dimer is present in the crystal but can not be identified in the electron density map Although crystals grew under many more conditions, crystals diffracting to reasonable resolution were only obtained in the cases reported here. This result is most likely due to the fact that in these cases, crystal contacts trap the G domains in defined orientations , whereasry mates , while iry mates . Crystal+-stimulated GTPase activity with and without attached MTSSL-label. No impairment of GTPase activity in comparison to wild type could be observed by the mutation itself or the introduction of the spin label  that the labeled position is located in the switch II region, which is flexible in the free and GDP-bound states, in line with the X-ray results, or (ii) that the spin label side chains are not restricted in their conformational space and populate multiple rotamers, or (iii) a combination of (i) and (ii). A general continuum of G domain orientations can be excluded from the results for positions E287R1 and D366R1. Control measurements of K+-stimulated GTPase activity  by Tikhonov regularization did not allow discrimination between a continuum of distances ranging from 25 Å to 50 Å with increasing probabilities for larger distances (shown in dark colours) or three to four distinct distances corresponding to different protein and/or spin label conformers . To clarify this issue, we additionally fitted the GDP data with a Monte Carlo/SIMPLEX algorithm assuming a sum of Gaussian-distributed conformers contributing to the dipolar evolution data  [41]. Thactivity  make sevx-complexed G domain dimer, the Cβ-Cβ distance of the S278- and E287-pair are 18 Å and 28 Å, respectively and thus somewhat shorter as compared to the GppNHp DEER data . However the MTSSL-side chain itself has an average length of 7 Å between the nitroxyl-radical and the Cβ-atom In the presence of the nonhydrolizable GTP analogue guanosine 5′-imidotriphosphate (GppNHp) the distance distributions comprise two fractions with different interspin distances for all three labeled positions. One larger distance  corresponds to the open state of the G domains as observed for the nucleotide-free and GDP-bound forms, whereas the other distance, contributing about 30% to the distance distribution  is characterized by significantly shorter distances , clearly indicating the presence of a second conformation, where the two G domains are in close proximity. As for the GDP-bound state, the GppNHp data for S278R1 were additionally fitted assuming a sum of Gaussian distributions. Despite differences especially in the distribution width for the two populations, this approach also reveals the presence of the two conformations of the G domains. In the X-ray structure of the AlFx, S278R1 and E287R1 show a single population maximum, with defined distances of 28 Å and 36 Å, respectively, in line with a closed conformation , with deviations due to spin label conformations as discussed above. Compared to the distances characterizing the closed conformation in the presence of GppNHp, the distance distributions for the transition state mimic are sharper and the maxima are slightly shifted. For E287R1 it decreases by about 1–2 Å and for position S278R1 the broad distribution between 20 and 30 Å converts to a more defined but asymmetric distribution with a major distance of 28 Å, which is well reproduced also by the Monte Carlo approach  does not show any significant differences concerning the major population in the distance distribution with an average distance of 29 Å for both nucleotide states  such as Rb+ (1.52 Å) and, to a lesser extent, NH4+ (1.44 Å), whereas Na+ (0.99 Å) and Cs+ (1.67 Å) do not show this effect + and NH4+ were also found to be coordinated to the K+ binding site in two MnmE G domain dimer structures GDP complexed with AlFx (pdb 2GJ9 and 2GJA) x, and GppNHp bound state in the presence of various cations, i.e., Na+, K+, Rb+, Cs+, and for S278R1 additionally in the presence of NH4+ for the GDP and GDP-AlFx state  spectroscopy to study the behavior of the G domains in full-length MnmE in different steps of the GTPase cycle. We were able to solve the first X-ray structures of full-length MnmE in complex with nucleotides. The structures confirm the previously postulated homodimer model + and triphosphate or a transition state mimic to induce G domain dimerization, the structures show the G domains in an open state, suggesting that the closed state is not stable under crystallization conditions. We can demonstrate indeed using a fluorometric assay with mant-GDP, that close juxtaposition of G domains with AlFx is inhibited in the presence of crystallization precipitants.Although the crystals for all structures presented here were grown in the presence of Kx and K+ is capable of stabilizing the closed state, and that this effect is specific, since the effect is absent with Na+ and Cs+ and is smaller with similar size cations such as Rb+ and NH4+.The interspin distances between the spin labeled G domains obtained by DEER directly prove for the first time that the G domains contact each other in the presence of triphosphate or transition state analogs. A notable feature of the GppNHp-bound state is the coexistence of an open and closed state, pointing out that a triphosphate analog is not sufficient to fully stabilize the closed state. However, recently a stabilizing effect of GidA on the closed state of the G domains was shown, indicating that regulation of the MnmE G domain cycle is coupled to other components of the tRNA-modification system In summary, we were able to directly demonstrate the conformational changes MnmE was suggested to undergo during its GTPase cycle MnmE forms a heterotetrameric complex with GidA C. tepidum and Nostoc sp. 7120 MnmE  were cloned into pET14b (Novagene) and expressed as N-terminal His-tagged proteins in Escherichia coli BL21-DE3. Cells were lysed in 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2 ( = buffer A) with 20 mM imidazole, 5 mM β-mercaptoethanol, 150 µM PMSF, and the proteins were purified by Ni-NTA, thrombin-cleavage of the His-Tag, and gel filtration on Superdex 200 in buffer A with 5 mM dithioerythritol (DTE). Cloning, expression, and purification of E. coli MnmE and mutants and preparation of nucleotide-free MnmE was carried out as described elsewhere 2, 5 mM DTE (buffer B) plus 5 mM GDP, 5 mM AlCl3, 50 mM NaF, and precipitant  were mixed. After 3 d the reservoir was changed to 100 mM Tris-HCl (pH 8.5), 2.250 M NaCl, 30% PEG 6000, and equilibrated for 2 more days. Crystals were soaked with precipitant supplemented with 12% glycerol and 5 mM 5-F-THF for 30 min and flash-frozen in liquid nitrogen. For NoMnmE·GDP, 1 µl of 20 mg/ml protein in buffer B with 5 mM GDP, 5 mM AlCl3, 50 mM NaF, and precipitant  were mixed and grown at 20°C. After 2 d crystals were cryo-dipped into reservoir solution with 28% (w/v) PEG 550 MME and flash-frozen into liquid nitrogen. For CtMnmE·GppCp, 40 mg/ml nucleotide free protein in buffer B with 5 mM GppCp was mixed (1∶1) with 100 mM MES, 46 mM NaOH, 12% PEG 4000, 40 mM NaCl, and crystals were grown at 20°C. After 2–3 d, crystals were flash-frozen in reservoir containing 20% glycerol. All datasets were collected at 100 K on beamline PX2  at wavelengths of 0.98003, 0.9796, and 1.28186 Å (Zn2+-edge) for CtMnmE·GDP, CtMnmE·GppCp, and NoMnmE·GDP, respectively. All datasets were processed, indexed, and scaled with XDS Crystals were obtained by hanging-drop vapour diffusion. For CtMnmE·GDP crystals, 1 µl each of 50 mg/ml protein in 50 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgClT. maritima MnmE (pdb 1XZP) with MOLREP www.pymol.org). For NoMnmE·GDP, Zn2+ atom positions were located by their anomalous signal. For CtMnmE·GppCp, a positive peak in the FO-FC-map close to the β- and γ-phosphate in the nucleotide binding site of G domain A was assigned to Mg2+, on the basis of its position at the usual Mg2+-site in G protein structures. Structures were analyzed by PROCHECK Initial phases were obtained by molecular replacement with the N-terminal and the helical domain of E. coli MnmE loaded with 0.5 µM of mGDP were incubated in 50 mM TriS-HCl (pH 7.5), 100 mM KCl (or NaCl), 5 mM MgCl2, 10 mM NaF with or without the precipitants 15% PEG 6000, 2,250 mM NaCl, or both or 22% PEG 550 MME at 20°C. The fluorescence of mGDP bound to MnmE, excited at 366 nm and detected at 450 nm, was monitored over time in a Fluoromax 2 spectralfluorimeter (Spex Industries). To initiate AlFx-complex formation, 1 mM AlCl3 was added and the fluorescence was continuously monitored. For analysis, fluorescence amplitudes were normalized to the amplitude before addition of AlCl3.10 µM of nucleotide-free E. coli MnmE-C451S , 100 mM KCl, 5 mM MgCl4Cl), 50 mM Tris-HCl, 5 mM MgCl2 (pH 7.4) with 5% (v/v) ethylene glycol (for H2O buffer) or 12.5% (v/v) glycerol-d8 (for D2O buffer), and 1 mM GDP, 1 mM GppNHp or 1 mM GDP, 1 mM AlCl3, 10 mM NaF, respectively.Pulse EPR experiments (DEER) were accomplished at X-band frequencies (9.3–9.4 GHz) with a Bruker Elexsys 580 spectrometer equipped with a Bruker Flexline split-ring resonator ER 4118X-MS3 and a continuous flow helium cryostat  controlled by an Oxford Intelligent temperature controller ITC 503S. Buffer conditions for the EPR experiments were 200–500 µM protein in 100 mM KCl : Coordinates und structure factors have been deposited with accession codes 3GEE (CtMnmE·GDP), 3GEI (CtMnmE·GppCp), and 3GEH (NoMnmE·GDP).Protein Data Bank (PDB)  Stereo image of the N-terminal domains of NoMnmE·GDP with the two bound 5-F-THF molecules and the 2FO-FC-map contoured at 2σ around the 5-F-THFs. (B) N-terminal domains of CtMnmE·GDP with the two bound 5-F-THF molecules and the 2FO-FC-map contoured at 2σ around the 5-F-THFs. (C–E) The bound nucleotide in the G domain of the structures CtMnmE·GDP (C), NoMnmE·GDP (D), and CtMnmE·GppCp (E) with the P-loop, the G-4-, and the G-5-mofiv O-FC-omit-maps as green meshes, contoured at 3σ  and 4σ (D). (C) GDP-bound to CtMnmE. (D) GDP bound to NoMnmE with the Zn2+-ion and its anomalous map contoured at 3σ (purple mesh). (E) GppCp bound to CtMnmE. Additionally the FO-FC-map at the β- and γ-phosphate calculated after fitting in GppCp, contoured at 2.5σ (purple mesh) is shown. On the basis of structural knowledge of the nucleotide binding site of G proteins, this peak in the FO-FC-map was assigned to Mg2+.(6.11 MB TIF)Click here for additional data file.Figure S2x-complex formation in the presence of precipitants used for crystallizationFluorometric assessment of G domain dimerization upon AlF. Normalized fluorescence amplitudes as a functions of time of the fluorescence labeled GDP analogon mGDP bound to MnmE in the presence of K+ as positive control (red curve) or Na+ as negative control (black curve) and NaF plus the respective precipitants together with K+, as indicated. At the beginning of the gap in the time traces, AlCl3 was added to initiate AlFx-complex formation and G domain dimerization, which only occurs in the presence of K+ and leads to an increase in fluorescence and which is impaired in the presence of various precipitants and K+ or when K+ is replaced by Na+.(1.29 MB TIF)Click here for additional data file.Figure S3Different orientations of the G domains. Superimposition of the five available MnmE homodimer structures CtMnmE·GDP, NoMnmE·GDP, CtMnmE·GppCp dimer A and dimer B, T. maritima MnmE dimer model generated with pdb 1XZP (TmMnmE) in ribbon representation with the N-terminal and helical domains in grey and the G domains  colored according to legend.(5.16 MB TIF)Click here for additional data file.Figure S4Stabilization of the G domains by crystal contacts. (A) Section of the crystal lattice of NoMnmE·GDP with close-up inset of the Zn2+-ion involved in crystal packing. In the crystal packing interface of the G domain to the symmetry related molecule, a Zn2+-ion , is complexed by side chain residues (shown as sticks) of the G domain and the helical domain of the symmetry related molecule. MnmE molecules are displayed as ribbon models in different colors. (B) Section of the crystal lattice of CtMnmE·GDP with MnmE molecules displayed as ribbon models in different colors. Two MnmE dimers are packed upside-down on each other with a toothing arrangement of the G domains.(4.25 MB TIF)Click here for additional data file.Table S1Rates vapp of K+-stimulated GTP-hydrolysis for wild-type MnmE, nonlabelled and MTSSL-labelled (denoted with R1) mutant MnmE proteins.(0.03 MB DOC)Click here for additional data file.Video S1The mobile MnmE G domains. The video shows sequentially the superimposed five available MnmE homodimer structures CtMnmE·GDP, NoMnmE·GDP, CtMnmE·GppCp dimers of molecules A and B, T. maritima MnmE dimer model generated with pdb 1XZP (TmMnmE) in ribbon representation with the N-terminal domains in blue, the helical domains in green and the G domains in red. The sequence in the video is: CtMnmE·GDP, CtMnmE·GppCp dimer A, CtMnmE·GppCp dimer B, TmMnmE, NoMnmE·GDP, TmMnmE, CtMnmE·GppCp dimer B, CtMnmE·GppCp dimer A. The video player may be set to playmode “infinite loop.”(1.93 MB MOV)Click here for additional data file.


Bovine herpesvirus type-1 (BHV-1) is an important pathogen of cattle that presents with a variety of clinical signs, including the upper respiratory tract infection infectious bovine rhinotracheitis (IBR). A seroepidemiological study of BHV-1 antibodies was conducted in England from 2002 – 2004: 29,782 blood samples were taken from 15,736 cattle from 114 herds which were visited on up to three occasions. Antibody concentration was measured using a commercial ELISA. Farm management information was collected using an interview questionnaire, and herd size and cattle movements were obtained from the cattle tuberculosis testing database and the British Cattle Movement Service. Hierarchical statistical models were used to investigate associations between cattle and herd variables and the continuous outcome percentage positive (PP) values from the ELISA test in unvaccinated herds.There were 7 vaccinated herds, all with at least one seropositive bovine. In unvaccinated herds 83.2% had at least one BHV-1 seropositive bovine, and the mean cattle and herd BHV-1 seroprevalence were 42.5% and 43.1% respectively. There were positive associations between PP value, age, herd size, presence of dairy cattle. Adult cattle in herds with grower cattle had lower PP values than those in herds without grower cattle. Purchased cattle had significantly lower PP values than homebred cattle, whereas cattle in herds that were totally restocked after the foot-and-mouth epidemic in 2001 had significantly higher PP values than those in continuously stocked herds. Samples taken in spring and summer had significantly lower PP values than those taken in winter, whereas those taken in autumn had significantly higher PP values than those taken in winter. The risks estimated from a logistic regression model with a binary outcome (seropositive yes/no) were similar.The prevalence of BHV-1 seropositivity in cattle and herds has increased since the 1970s. Although the study population prevalence of BHV-1 was temporally stable during study period, the associations between serological status and cattle age, herd size, herd type, presence of young stock and restocked versus continuously stocked herds indicate that there is heterogeneity between herds and so potential for further spread of BHV-1 within and between herds. Herpesviridae, subfamily Alphaherpesvirinae. It is an important pathogen of cattle worldwide [Bovine herpesvirus type-1 (BHV-1) is a member of the family orldwide . ConventBHV-1 generally infects cattle greater than 6 months of age once maternal immunity has waned . ClinicaBHV-1 has been in Great Britain (GB) since the 1960s ,8, but wThe reported risk factors for the presence of BHV-1 antibodies in cattle in the Netherlands included a large herd size, dairy herds with beef/veal cattle, a high density of herds in the municipality , purchasThis paper presents the largest longitudinal study of BHV-1 seropositivity in cattle in the UK, and its association with cattle age, birthplace, herd type and size.The data used in this paper came from 114 dairy and/or suckler herds in south west England. Farms were visited between 2002 and 2006 and were located within the Randomised Badger Culling Trial (RBCT) . In addiSamples of blood (up to 10 ml) were collected under Home Office licence from all accessible cattle over 2 years of age at three routine visits, at approximately one year intervals. A subset of herds (n = 15) had a whole herd test, i.e. blood samples were collected from cattle of all ages, prompted by the presence of an animal persistently infected (PI) with bovine viral diarrhoea virus (BVDV). Four herds had a whole herd test instead of a routine third visit; otherwise whole herd tests were additional to routine tests.All blood samples were centrifuged at the University of Warwick at 3220 g for 15 minutes, and serum was removed and frozen at -20°C.th June 2003 and 18th February 2004 to obtain information on clinical signs of infectious bovine rhinotracheitis (IBR) disease and use of a BHV-1 vaccine. Four out of 114 farmers did not complete the questionnaire.Farmers completed an interview questionnaire between 17At each visit, the unique identity of cattle was recorded from their ear tag and linked to the relevant blood sample. When appropriate, freeze brand numbers were also recorded. The ear tag was matched to the individual cattle data on the British Cattle Movement Service (BCMS) to obtain/confirm the following: the date of birth, whether the animal was homebred or purchased, breed and sex. More than 99% of cattle were matched within the BCMS dataset. The main reason that cattle were not matched was because they were born before 2001, when it was not compulsory to record cattle birth dates. Herd size was estimated from the cattle tuberculosis testing data (VetNet database), and validated during farm visits.All data were entered into a relational database  using Microsoft Access (Microsoft Corp. US) as a front end. All data were screened for errors. When data mismatches were detected, data were re-checked to determine the source of the mismatch and where possible this was corrected.1) was detected using the SVANOVIR® IBR-Ab  indirect enzyme-linked immunosorbent assay (ELISA). All testing was completed according to the kit instructions. Internal controls were included on each plate to control for batch to batch variation. All samples were run in duplicate, and all testing was completed at the University of Warwick. Positive samples were re-tested when the duplicates were more than 0.25 optical densities (OD) units apart or when the OD of the positive controls was more than 0.2 OD units apart. The sensitivity and specificity of the serological kit to the serum neutralisation test have been reported as 97.4% and 92.4% respectively [The presence of BHV-1 specific antibodies  were used. Dataset A included all serological results for all cattle from herds that were not vaccinated against BHV-1 for all visits . Dataset B was a subset of Dataset A and included all serological results from cattle >2 years of age from the three routine visits and 4 whole herd visits .A herd was defined as BHV-1 seropositive when at least one cow in the herd was above the cut off for BHV-1 on at least one occasion.Cattle were defined as BHV-1 seropositive when above the cut off for BHV-1 at least once.The herd BHV-1 seroprevalence was calculated at each visit as the number of seropositive cattle in the herd divided by the total number of cattle tested.Seroconversion was defined in a herd when at least one cow changed from being seronegative to seropositive between two visits.Two outcomes were modelled to investigate associations between BHV-1 seropositivity and herd and cow variables. These were the continuous outcome PP, and a binary (positive/negative) outcome (seropositive yes/no).The variables listed in Table Associations between the outcomes and herd and animal factors were initially screened using Mann-Whitney and chi-squared tests. Variables with a significance value of <0.20 in the univariate analysis were tested in two multi-level models: one with a continuous outcome of PP value and one with a binary outcome of seropositive yes/no. There were three hierarchical levels in the models: repeated measure at each visit (level 1), clustered by cow (level 2), and herd (level 3) to control for the dependency between repeated measures within cattle and cattle within herds. The multivariable models were built using both manual forward selection and backwards elimination.The models took the form:Yijk is the PP value or seropositivity value at visit i from cow j in herd k. βX0 is the intercept, βX is a series of vectors of fixed effect varying at the herd (k), cattle (jk) and visit (ijk), νk + ujk are the variances at the herd and cattle levels, and eijk is the residual variance.where The model fit was assessed by plotting the raw, standardised and deletion residuals, and the leverage and influence for each random effect and all fixed effects were also assessed . The detCorrelations between the explanatory variables were investigated using chi-squared tests. Variables that were significantly (P < 0.01) associated with each other were noted to help with interpretation. All analyses were done using MLwiN version 2.02  .All 107 unvaccinated herds were visited at least once, 96 were visited twice and 95 were visited on three occasions, generating 9136, 8099 and 7850 serum samples, respectively. A further 1833 blood samples were collected from additional visits or because of a whole herd test and/or cattle were under 2 years of age.The median herd size of vaccinated herds was 274; in contrast, the median herd size of unvaccinated herds was 185. The median herd size of unvaccinated seronegative and seropositive herds was 58 and 193, respectively. Nine percent (10/114) of farmers reported that they had seen clinical signs of IBR in their herd in the past, and two of these farmers also reported that they now vaccinated their cattle. All vaccinated herds had at least one seropositive animal compared with 83.2% of unvaccinated herds with at least one seropositive animal. The mean within-herd seroprevalence in vaccinated herds was 74.9% compared with 43.2% in unvaccinated herds.U = 1908; P <0.05, (Mann-Whitney U-test).There were 89 herds  with at least one BHV-1 seropositive bovine on at least one occasion .The seroprevalence of antibodies against BHV-1 increased with age Figure . There wχ2 = 12137, P < 0.01. There was a significant association between season of visit and PP values. Samples taken in the spring and summer had significantly lower PP values than samples taken in winter; whereas those taken in autumn had significantly higher PP values than those taken in winter.Of the 3,452 cattle with no data on their place of birth, 2,101 (60.9%) were seropositive. Of the 10,791 cattle with data, purchased cattle were significantly more likely to be seropositive (54.1%) than homebred cattle (43.6%). The mean PP value was higher for cattle purchased into dairy herds, and increased at a greater rate over time from purchase in cattle ≥ 2 years of age at purchase , from 10,039 cattle level 2) in 102 herds (level 3) with no missing values. In the continuous outcome model there was a significant increase in PP value with age. Cattle from herds with >200 cattle had higher PP values compared with those from herds with ≤ 200 cattle, and there were higher PP values in herds that were formed by restocking after the FMD epidemic of 2001, compared with herds that were continuously stocked. Herds with dairy cattle had higher PP values compared with those without dairy cattle. Herds with grower cattle had lower PP values compared with those without grower cattle Table . In cont in 102 hThe sum of the level 1, level 2, and level 3 variances was 0.28. The greatest unexplained variance in PP values in the final multi-level model was between cattle, making up 48% of the total variance. However, the variances were significant at all three levels  was fitted Table . The varThis is the largest seroepidemiological study of BHV-1 carried out in cattle herds in the UK, and the first for more than 15 years. The seroprevalence of BHV-1 has increased dramatically since the 1960s in both cattle and herds. In the 1960s the seroprevalence in cattle was 2.1% , and 83.A particularly interesting result is the higher seroprevalence in herds that were restocked after FMD. Carrique-Mas et al. (2008)  reportedThe seroprevalence of BHV-1 increased with herd size in the univariate analysis and in the hierarchical model; this is consistent with other studies ,26-28. TThe age serological profile is consistent with life-long seropositivity. Antibodies to BHV-1 in calves of ≤ 6 months of age probably correspond to colostrum derived maternal immunity. This wanes by 1 year of age, and starts to rise by 2 years of age because of active infection. This suggests that the optimum age for vaccination of cattle is 10 – 14 months of age, although with some active seroconversion observed in adult cattle and a seroprevalence only exceeding 50% in older animals, vaccination is likely to have a positive impact when administered at any age. All the vaccinated herds had seropositive cattle, while approximately 80% of the unvaccinated herds had seropositive cattle. On average, less than 50% of the cattle in the unvaccinated herds tested positive at each visit, whilst >75% of the cattle from vaccinated herds were positive. These data suggest that either some cattle in vaccinated herds were not vaccinated, that vaccination does not always induce antibody or that antibody titre induced by vaccination wanes over time. It is possible that antibody titre generated by natural infection also wanes over time, but there is no previous work on this hypothesis.In the current study, when the herd was a dairy herd rather than a suckler herd, the rate of seroconversion in adults was greater. The average age of cattle did not differ between dairy and suckler herds, and so this is a true effect, possibly due to winter housing or other stressors related to dairy cow management.Housing and winter stressors are also an explanation for the small but significant effect of season on PP values. However, 31% and 47% of visits were 11–13 months apart for routine visits 1 and 2 and routine visits 2 and 3, respectively, so that there was a tendency for the same herds to be sampled in the same season and so herd effect is interpreted as an apparent seasonal effect. Other explanations are that seasonal culling and replacement of cattle might influence herd prevalence of BHV-1 antibodies.All the variables above became non-significant when the binary variable seroconversion (indicating whether seroconversion was observed in the herd) was introduced into the final model. This indicates that the model risk factors are those associated with virus circulation between cattle on the farm.The use of different modelling approaches has been discussed elsewhere ,30 but nIn the discrete model, we assumed that the serological measurement of seropositive/seronegative had perfect sensitivity and specificity. It is possible that bias could be introduced into the analysis if the sensitivity or specificity of the test varied with other variables, for example, with cattle age, or time since infection, however this has not been reported for BHV-1 antibody tests. The continuous outcome (PP) calculated from the ELISA will have had some random error Figure , but avoThe results of this study do not necessarily represent all cattle herds in England, because of the locality of the farms sampled and because the analysis was based primarily on the serological values for BHV-1 from adult cattle. Using only unvaccinated herds in the study may have reduced the extrapolation of the results, because these herds may have had different management practices from vaccinated herds, but there were only 7 herds that reportedly used vaccination and so analysis of these herds' managements was not possible. In addition, there might have been an overestimation of the infection derived seroprevalence because purchased cattle might have been sourced from a vaccinated herd and so had antibodies derived from vaccination: we do not know the vaccination status of source herds. The serological test used in the current study did not distinguish between vaccine derived and pathogen derived antibody so this was unavoidable. It is interesting that only 7 herds were vaccinated and that farmers reported little clinical disease from BHV-1, suggesting that this infection is not an obvious production constraint or clinical disorder in most herds.Finally, in the current analysis we could not distinguish between BHV-1 sub-types, the epidemiology of individual types might be different and this might account for some of the unexplained heterogeneity. BHV-1 subtype 3 is now classified as a new species (bovine herpesvirus-5 or BHV-5) . It is pIn conclusion, the level of BHV-1 infection has increased dramatically since previous estimates from the UK, with approximately 83% of the unvaccinated herds in the south west of England seropositive. This study adds to previous work with the finding that infection might have reached a temporal but not a spatial equilibrium and that between herd variability in PP might arise through variability in cattle age, herd size, herd purpose and the formation of new herds.KAW helped collect samples, performed the data analysis and drafted the manuscript. GFM and LEG are the funded co-applicants who designed the study, supervised data collection and analysis and finalised the manuscript. SJM coordinated and completed ELISA testing. ARV assisted in design of the questionnaire and coordinated data collection and supervised collection of blood samples. SM setup and maintained the study database and downloads. All authors read and approved the final manuscript.


Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases.Cloning of the Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed.Comprehensive in silico analyses indicate that the recently identified The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. Biochemical and molecular studies amply demonstrated that plants express a multitude of carbohydrate-binding proteins  ,2. Thougin situ with conspecific N-glycosylated nuclear proteins [Though applicable to the majority of all previously studied plant lectins, novel concepts had to be developed after the identification of several novel hormone or stress-responsive lectins. By virtue of their subcellular location and specificity this new class of lectins is at least in principle capable of interacting with endogenous receptors in the cytoplasmic/nuclear compartment of the plant cell ,5-7. Basproteins . Even inproteins -14. Howeproteins .in silico analysis of the family of cytoplasmic/nuclear protein(s) comprising domain(s) equivalent to the recently cloned Euonymus europaeus agglutinin [Euonymus lectin) domain(s) are expressed in all Embryophyta - ranging from liverworts to flowering plants - for which a reasonable number of sequences has been deposited, but could not be found in any other eukaryote or prokaryote hitherto. Despite the EUL domain itself being fairly well conserved, the holoproteins comprising such (a) domain(s) exhibit a marked structural heterogeneity. Some proteins consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. Both the N-terminal domain and the linker sequence are highly variable. Transcriptome/genome analyses revealed that some species express a single EUL per diploid genome whereas up to eight structurally different proteins are found in others. Furthermore, expression analyses revealed that EUL domains are present in many stress response proteins suggesting a role of this lectin domain in stress signalling. The identification of the EUL family is discussed in view of the increasing importance of glycobiology in plant cell biology in general, and the understanding of the physiological role of plant lectins in particular.Here we report the identification and glutinin . The maiEuonymus europaeus agglutinin (EEA) cannot be assigned to one of the existing lectin families [Euonymus agglutinin [e-05. Due to redundant annotations the number of (putative) proteins is considerably lower (approximately 50). Of all these entries only EEA has been purified and characterized. For a few others (like the rice OSR40 proteins) there is experimental evidence based on protein analysis techniques that they are actually expressed. All other hits detected by BLASTp searches refer to hypothetical proteins the sequence of which is deduced from either cDNA or genomic sequences. At present there is no uniform naming for all these putative proteins. Most of them are still annotated as "putative/hypothetical protein", "expressed" protein, "unknown" protein or "stress-responsive" protein.A recent reinvestigation using a molecular approach revealed that the families  but sharfamilies . Based oglutinin  (AdditioTulipa gesneriana) bulbs  [Arabidopsis thaliana (At2 g39050) expressed in Pichia pastoris agglutinates rabbit erythrocytes  and hence must be capable of interacting with carbohydrate structures present in the erythrocyte membrane. The obvious carbohydrate-binding activity of three different EUL proteins from taxonomically unrelated species strongly indicates that several EUL domains can be considered lectin domains. It should be emphasized, however, that sequence similarity to the Euonymus lectin does not necessarily imply that a given domain possesses carbohydrate-binding activity.Several experimental data unambiguously demonstrate that the EUL domain represents a new carbohydrate-binding domain. First, glycan array binding studies showed that EEA has high affinity towards blood group B oligosaccharides, but also binds to high mannose N-glycans . Second,d TxLMI)  that untEuonymus lectin domain represents the structural unit for a novel lectin family ubiquitous in terrestrial plants (Embryophyta). Since neither the rice OSR40 proteins nor the putative homologs found in other plants can be classified under the ricin B family, a more appropriate nomenclature should be introduced. Taking into account that the Euonymus agglutinin was the first identified member and, in addition, possesses a well-defined biological activity (in casu carbohydrate-binding), it seems logical to name this novel protein family after the Euonymus agglutinin and consider the Euonymus lectin domain as the diagnostic structural unit. Accordingly, the term 'Euonymus lectin domain' (or 'EUL domain') will be adopted for all structural units equivalent to the Euonymus lectin subunit, and the term 'EUL family' will be used to refer to the group of proteins containing at least one EUL domain.Transcriptome analyses showed that virtually all Embryophyta species for which a reasonable number of sequences were deposited express one or more proteins with a domain similar to the EEA polypeptide (see below). Therefore, it appears that the To the best of our knowledge the acronym EUL is not used yet for any protein family or conserved protein domain, and hence can be introduced without the risk of confusion. The terms 'EUL' and 'EUL family' are preferred to 'OSR40' and 'OSR40 proteins' for two reasons. First, the name 'OSR40' includes no information about the biological activity of the domain. Second, the term 'OSR40' does not refer to a well-defined structural unit but to a 40 kDa ABA/salt stress responsive rice protein with a complex structure . Hence, the term 'OSR40' is inappropriate to refer to all related proteins that include only a single 150 AA domain and differ in molecular mass.Euonymus lectin - exclusively of a sole EUL domain. However, in most cases the EUL domain is preceded by an unrelated N-terminal domain varying in length and composition/sequence. In a minority of the single-domain EULs a signal peptide was detected at the N-terminus, indicating that some vacuolar EULs presumably also exist. In addition to an N-terminal domain, an extra domain at the C-terminus which can also differ in length is found in a few single-domain EULs. Most but not all two-domain proteins contain an extra N-terminal sequence and a linker between the two EUL domains. Despite differences in the length of the N-terminal sequences as well as the linkers, the subfamily of two-domain EULs is less heterogeneous than that of the single-domain proteins.Comprehensive sequence analyses demonstrated marked differences in the overall structure of proteins with EUL domain(s). Based on the sequence information available at present, seven types of proteins containing a single EUL domain and five types of proteins with two EUL domains can be distinguished Figure . Table 1The obvious differences in domain architecture between different members of the EUL family combined with the simultaneous occurrence of multiple structurally different EULs in some species necessitate a consistent nomenclature. Therefore, a classification system was elaborated based on the overall domain architecture and the length/sequence peculiarities of the accessory domains. Individual proteins containing an EUL domain are indicated by a species code, composed of the three first characters of the genus name and the first two characters of the species name, followed by one of the 12 domain architecture codes  complete genome sequences are available Figure .Arabidopsis thaliana genome harbours a single EUL gene/protein (At2 g39050). According to both the TIGR and TAIR annotations At2 g39050 belongs to a 'Hydroxyproline-rich glycoprotein family' and contains a 'QXW lectin repeat domain' (Pfam: PF00652 = Ricin-type beta-trefoil lectin domain), whereas in the MIPS database the protein is annotated as 'similar to stress responsive lectin-like cDNAs from rice'. However, it is evident now that the C-terminal part of this Arabidopsis protein corresponds to an EUL domain and accordingly should be classified in the EUL family. The 154 AA EUL domain in this Arabidopsis protein shows 44% sequence identity and 71% sequence similarity to the EUL domain of EEA. In addition, this protein contains a long (approximately 160 residues) unrelated N-terminal domain that shares no significant similarity with any other known domain. Due to the presence of this long N-terminal domain the Arabidopsis EUL is classified as a type S3 EUL (ArathEULS3).The ArathEULS3 gene were also identified in the completely sequenced genomes of Medicago truncatula (barrel clover), Vitis vinifera (common grape vine), Ricinus communis (castor bean) and Carica papaya (papaya). The corresponding proteins are expressed and have the same domain architecture as ArathEULS3  that are located at different loci and according to transcriptome data are expressed. In addition, a third gene (GlymaEULS3C) tandemly arrayed to GlymaEULS3B could be identified that encodes an EUL protein with a shorter N-terminal domain . The occurrence of GlymaEULS3A and GlymaEULS3B can be explained by the fact that soybean is a "diploidized tetraploid", whereas GlymaEULS3C most probably results from an in tandem duplication. At present, no similar in tandem arrayed pair of EUL genes was identified in any other dicot. However, as described below, in tandem duplication made an important contribution to the evolution of EUL genes in grasses (and perhaps in other monocots as well).The genome of soybean comprises two genuine orthologs of the n Figure . No corret al. [In 1995, Moons et al.  identifiet al. . TherefoBLAST searches against the completed rice genome confirmed the occurrence of an EUL gene family. Nine genes could be identified that encode proteins comprising one or two EUL domains. Expression was detected for only five of these genes Figure  suggestiArabidopsis-type EUL(s) genes OrysaEULS0A and OrysaEULD0, on chromosome 3; the two non-expressed (pseudo)genes OrysaEULS0B and OrysaEULS0C as a cluster on chromosome 12.Based on its overall domain structure OrysaEULS3 can be considered a genuine ortholog of the O. sativa, it is suggested that the OrysaEULS0B and OrysaEULS0C genes might be pseudogenes in O. sativa. Nonetheless this conclusion cannot be extrapolated to all Oryza species. A cDNA sequence encoding an S0 type EUL was deposited, indeed, for O. punctata. Interestingly, a virtually identical nucleotide sequence can be assembled from the O. sativa genomic sequence by joining the first exon of Os12 g08340 and the second exon of Os12 g08310. A closer examination shows that the genomic sequences covering Os12 g08340 and Os12 g08310 contain the coding sequence of an S0 type EUL protein (as expressed in O. punctata) in which the exons encoding the N- and C-terminal part are interrupted by a very long intron  that apparently comprises a transposon. This might indicate that the O. sativa gene encoding a S0 type EUL protein was - in evolutionary terms - recently inactivated through the insertion of a transposon.Because no trace of type S0 EUL expression could be detected in Sorghum bicolor expresses closely related orthologs of all five EUL proteins expressed in O. sativa   EUL genes that are not found in the rice genome. First, there is a third two-domain protein (SorbiEULD1C) for which no ortholog could be identified in rice. Second, the genome apparently contains four genes (SorbiSv1-4) encoding single domain EUL proteins that are synthesized with a signal peptide. Though the exact subcellular location of these proteins is not known, it seems evident that they are synthesized in the ER and follow the secretory pathway. To distinguish them from the 'cytoplasmic' EUL they are referred to as 'vacuolar' EULs. For both SorbiSv1 and SorbiSv2 corresponding ESTs could be retrieved indicating that these genes are expressed.BLAST searches in the genome and transcriptome databases indicated that Z. mays expresses orthologs of all five rice EUL proteins   and a single gene encoding a two-domain EUL protein (PhypaEULD1)  Figure . PerfectSelaginella moellendorffii  resulted in the identification of a complex set of at least 34 genes encoding EULs of eight different types: four different single-domain cytoplasmic types , one single-domain vacuolar type (SelmoEULSv), and three two-domain types   Figure . For eacS. moellendorffii genome contains three "novel" types of two-domain genes. Two genes (SelmoD0a-b) encode two-domain proteins without N-terminal domain and two other (SelmoD3a-b) two-domain EULs with a long N-terminal domain. Finally, the Selaginella genome contains at least 6 pairs of genes (SelmoD4A-Fa-b) encoding SelmoEULD4 proteins. SelmoEULD4 proteins are like SelmoEULD0 two-domain EULs without N-terminal domain. However, they are only distantly related to other two-domain EULs. ESTs have been identified for all types of proteins  except for SelmoEULS0, SelmoEULS1C, and for the vacuolar proteins.S4-type EULs resemble the S2- and S3-type proteins found in monocots and dicots but distinguish themselves by the presence of an extra 34 AA residue C-terminal domain. Moreover, the latter is located on a separate exon. Besides these "unique" S4-type genes the To further corroborate the presence and composition of the EUL gene complement in other plant species, a thorough analysis was performed of available transcriptome data Figure . A detaiMarchantia polymorpha. Both proteins comprise a short N-terminal domain followed by an EUL domain and a short to medium long C-terminal domain  and cycads (Cycas rumphii and 3 Zamia species). Conifers such as Pinus taeda (loblolly pine) also express a complex set of EUL proteins comprising S2, S3, D1 and D2-type proteins. Although the complement of EUL proteins expressed in Picea sitchensis (Sitka spruce) resembles that in P. taeda it is certainly not identical  and Magnoliids (e.g. Liriodendron tulipifera). Within Eudicotyledons EUL sequences are present in stem eudicotyledons, (e.g. Aquilegia formosa × Aquilegia pubescens) as well as in virtually all EST databases from core eudicotyledons. Most species express a single S3-type protein per diploid genome but several species  express complex mixtures of S3-type EUL proteins.Within flowering plants some ESTs encoding EUL proteins are found in basal Magnoliophyta (e.g. O. sativa.A search for EUL domains in Liliopsida (monocotyledons) revealed that all EST databases from monocots contain sequences encoding EUL domains. The whole of retrieved sequences indicates that most if not all of these monocots express a set of single-domain and two-domain proteins comparable to that found in Arabidopsis electronic fluorescent protein browser [Several cDNA and EST sequences have been identified that confirm the expression of the EUL homolog ArathEULS3 (At2 g39050) in Arabidopsis. Therefore the expression profile of this EUL homolog was studied using the  browser .th flower stage. The highest absolute fluorescence value in untreated plants (955) was observed in the sepals of flowers but cauline leaves also clearly show expression of ArathEULS3. Microarray expression analyses of leaf mesophyl cells and guard cells [The expression of ArathEULS3 is developmentally regulated with a high expression in senescent leaves and in flowers from the 15rd cells  revealedThe relative expression  of ArathEULS3 was studied for different abiotic as well as biotic stresses  as well as Bryophyta (mosses) and Marchantiophyta (liverworts). Comprehensive BLAST searches of the completed genomes (and annex transcriptome) of Chlamydomonas reinhardii, Chlorella sp., Micromonas pulsilla, Ostreococcus sp., and Volvox carteri yielded no significant hit, suggesting that the EUL domain is absent from these Chlorophyta. Thus, it seems likely that the EUL domain was developed/acquired after the separation of the Chlorophyta and Embryophyta lineages (approximately 500 million years ago).n family . DetaileAedes aegypti (an insect) whole larvae, Wuchereria bancrofti (a nematode) larvae, and Xenopus laevis whole embryos . The best guess is that the larvae used for the construction of the respective EST libraries were (indeliberately) contaminated by wind carried grass pollen grains that upon RNA extraction contributed to the EST library. Accordingly, all evidence suggests that the EUL domain was developed in plants rather than acquired by either vertical or horizontal inheritance from a prokaryotic ancestor. However, it can not be precluded that other yet unidentified organisms have developed in parallel the same protein domain.At present there is no evidence for the occurrence of proteins with EUL domain(s) in other eukaryotes  or prokaryotes. Hence, one can reasonably conclude that the EUL domain is confined to the Embryophyta. It should be noted here that a few ESTs with typical EUL sequence were also found in the transcriptome of ArathEULS3 contains three introns, one of which is located within the stop codon. The first exon comprises the N-terminal domain plus approximately the first 40 residues of the EUL domain whereas the rest of the EUL domain is divided over the second and third exon. A very similar exon/intron structure was also found in the genes expressing other EULS3 proteins though the length of the second intron can be much longer, as is the case in PoptrEULS3A and PoptrEULS3B where the second intron contains 1,397 and 3,480 nucleotides, respectively. We conclude that the position of the first and second intron in the EUL domain is conserved in all EULS3 genes of dicots. The third (first-order) intron is invariably located in the stop codon  have an intron/exon structure reminiscent to that of the EULS3 gene (four introns in the open reading frame and one in the stop codon). However in most proteins  some of these introns are apparently missing. Interestingly, all introns in the open reading frame are positioned within an EUL domain, and all expressed EUL genes contain a nearly identical exon sequence corresponding to the C-terminal part (comprising 76 or 77 amino acid residues) of the respective proteins. In the two-domain proteins of the lower plants S. moellendorfii and P. patens introns occur also in the N-terminal domain and/or between the linker and an EUL domain  encoding a single-domain S3 type EUL. Second, the genome of monocots comprises a complex family of genes which encode proteins with either one or two in tandem arrayed EUL domains. Only one of these genes is a genuine ortholog of the single-domain S3-type EUL genes in dicots. The single domain S3-type EULs are present in dicots, monocots and mosses, and are markedly conserved among these divergent classes. Moreover, they are encoded by genes with a strictly conserved intron/exon structure. Therefore the S3-type EUL can be considered a universal EUL. Third, (some) lower plants such as Selaginella have a more complex set of EULs. Furthermore, the spikemoss Selaginella and the liverwort Marchantia express some EUL types that could not be identified in higher plants, such as EULs with an additional C-terminal domain. From these observations it is hypothesized that the EULs of seed plants evolved from the EULs of lower plants. Additionally, in some EUL genes of lower plants introns are positioned in the N-terminal domain or in the linker, which is in strong contrast to genes encoding EULs from higher plants. Nonetheless, most genes encoding EULs do typically have an intron sequence in their stop codon. Next to cytoplasmic EULs which occur in all investigated plant species, vacuolar EULs were detected only in some monocots and in Selaginella.Though only 12 (nearly) completed genomes have been screened, several important conclusions can be drawn with respect to the composition of EUL genes/proteins in plants. First, the genomes of dicots contain only one or two genes  in this EUL domain are highly conserved  the EUL domain arose only once in the course of plant evolution, whereupon it could expand either by fusion with other segments or by gene duplication resulting in more complex EUL proteins; (2) the taxonomically most ancestral operational taxonomic units should make up the root of the tree. On these grounds SelmoEULS0, which is the oldest and simplest EUL, indeed, could be the sole outgroup member, but upon rooting with this single operational taxonomic unit it immediately became clear that the next most simple EULs (types S1 and Sv) of this species could likewise be assigned to the root partition (marked A) of the tree.Selaginella in this case), which are the most primitive vascular plants. Splitting off from the root of the tree there are two derived sister clusters: a smaller one  complete genomes are available using the following databases:EUL homology searches with the nucleotide as well as the protein sequence were done with the different BLAST programs  availablhttp://castorbean.tigr.org- The TIGR Castor bean database Selaginella genomics database http://selaginella.genomics.purdue.edu/; http://genome.jgi-psf.org/Selmo1/Selmo1.home.html- The Physcomitrella patens genome database- The http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.htmlhttp://www.cosmoss.org/- The moss computational biology toolbox Populus trichocarpa genome database- The http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.htmlVitis vinifera genome database- The http://www.cns.fr/spip/Vitis-vinifera-e.htmlhttp://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=rice- The TIGR Rice Genome Database Arabidopsis database http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=arab- The TIGR Arabidopsis thaliana TAIR database http://www.arabidopsis.org- The Arabidopsis thaliana MIPS database http://mips.gsf.de/proj/plant/jsf/athal/index.jsp- The Sorghum bicolor genome database http://www.phytozome.net/sorghum- The Medicago truncatula genome database http://medicago.org/genome/IMGAG- The http://maize.tigr.org- The TIGR Maize database http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=wheat- The TIGR Wheat genome database Glycine max genome database http://www.phytozome.net/soybean- The http://www.cbs.dtu.dk/services/SignalP[http://www.psort.org/. An electronic analysis of the expression profile of the EUL from Arabidopsis was performed using the Arabidopsis eFP Browser http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi[All retrieved sequences were analyzed individually. In the absence of complete coding sequences, contigs were reconstructed from ESTs showing overlaps of at least 200 identical nucleotides. Searches were completed on February 28, 2009. Introns in the genomic sequences were identified manually by comparison to the nucleotide sequences of corresponding ESTs or cDNAs. Signal peptides were predicted with the SignalP3 program s/SignalP and the fpWeb.cgi,25.http://www.ebi.ac.uk/clustalw/[2), with the three states in the Hidden Markov Model corresponding to a rate of change of 0.498 with probability 0.532 (representing more conserved positions), 1.472 with probability 0.442 (representing medium variable positions), and 3.190 with probability 0.027 (representing hypervariable positions). Second, the latter parameter settings - wherever applicable - were used for calculating Neighbor Joining trees (program protdist followed by neighbor) for comparative purposes in order to verify the consistency of phylogenetic inference. A third phylogenetic method was applied using the MrBayes program with mixed models [For phylogenetic analysis, an amino acid sequence dataset was compiled with one operational taxonomic unit for every single EUL domain from any known EUL protein. Sequences were aligned using ClustalW clustalw/. Multiplclustalw/. Two typclustalw/. First, d models ; where pd models  or Dendrd models .Euonymus europaeus agglutinin; EUL: Euonymus lectin.AA: amino acid; ABA: abscisic acid; EEA: EF carried out some of the analyses, prepared the tables and figures and the primary drafts of the manuscript and contributed to finalization of the text and journal-specific formatting. WP conceived the study, analyzed genomic sequence and EST sequence information and provided valuable editorial advice. TV and MO performed the phylogenetic analyses. EVD provided overall project leadership, supervised collection, analysis and interpretation of the data, and codeveloped interim and final drafts of the manuscript. All authors read and approved the final manuscript.Additional Figures S1-S5. Figure S1: Sequence alignment of EUL sequences with known lectin sequences.  Alignment of the amino acid sequences of EEA and the individual ricin-B domains of Ricinus communis agglutinin (AAA33869.1). (C) Alignment of the amino acid sequences of the EUL protein from Curcuma longa (CurloEULS3) and the N-terminal sequence and two tryptic peptides of the tulip lectin TxLMI. The N-terminal sequence of TxLMI is shown in bold. Figure S2: Multiple sequence alignment of the amino acid sequences of the EUL domain of Euonymus europaeus and the S3-type EULs from different plant species. The lectin abbreviations can be found in Additional file Figure S3: Expression profile of the EUL from Arabidopsis thaliana  based on the data provided by the Arabidopsis eFP browser. Relative expression of ArathEULS3 in the shoots of 18 day-old seedlings subjected to different abiotic stresses (A) and treatments with plant hormones (B). Relative expression of ArathEULS3 in leaves of 4 week-old plants after infection with the pathogens Botrytis cinerea (C), Pseudomonas syringae pv. tomato DC3000 (avirulent strain) and Pseudomonas syringae pv. tomato avrRpm1 (virulent strain) (D). Figure S4: Alignment of EST sequence from Aedes aegypti (Aedae) and a nearly identical sequence from creeping bentgrass (Agrostis stolonifera) (Agrst). Identical nucleotides are indicated by asterisks. Figure S5: Amino acid sequences of proteins containing one or two Euonymus lectin (EUL) domains. The EUL domains are shaded yellow and green. Signal peptides are shaded grey. Only the EUL domains were used for construction of the phylogenetic tree shown in Figure tinin AAA3869.1. - and double (D)-domain EULs used to construct a phylogenetic tree (Figure e Figure . SequencClick here for file


Methods that aim at predicting interaction partners are very likely to play an importantrole in the interpretation of genomic information. iSPOT (iSpecificity Prediction OfTarget) is a web tool (accessible at http://cbm.bio.uniroma2.it/iSPOT) developed for theprediction of protein-protein interaction mediated by families of peptide recognitionmodules. iSPOT accesses a database of position specific residue-residue interactionfrequencies for members of the SH3 and PDZ protein domain families. The softwareutilises this database to provide a score for any potential domain peptide interaction.        iSPOT: 1. evaluates the likelihood of the interaction between any of the peptidescontained in an input protein and a list of domains of the two different families; 2. searchesin the SWISS-PROT database for potential partners of a query domain; and 3. has accessto a repository of all the domain/target peptide interaction data.