Calling on a million minds for community annotation in WikiProteins

WikiProteins enables community annotation in a Wiki-based system. Extracts of major data sources have been fused into an editable environment that links out to the original sources. Data from community edits create automatic copies of the original data. Semantic technology captures concepts co-occurring in one sentence and thus potential factual statements. In addition, indirect associations between concepts have been calculated. We call on a 'million minds' to annotate a 'million concepts' and to collect facts from the literature with the reward of collaborative knowledge discovery. The system is available for beta testing at http://www.wikiprofessional.org.     

The molecular evolution of the vertebrate behavioural repertoire

How the sophisticated vertebrate behavioural repertoire evolved remains a major question in biology. The behavioural repertoire encompasses the set of individual behavioural components that an organism uses when adapting and responding to changes in its external world. Although unicellular organisms, invertebrates and vertebrates share simple reflex responses, the fundamental mechanisms that resulted in the complexity and sophistication that is characteristic of vertebrate behaviours have only recently been examined. A series of behavioural genetic experiments in mice and humans support a theory that posited the importance of synapse proteome expansion in generating complexity in the behavioural repertoire. Genome duplication events, approximately 550 Ma, produced expansion in the synapse proteome that resulted in increased complexity in synapse signalling mechanisms that regulate components of the behavioural repertoire. The experiments demonstrate the importance to behaviour of the gene duplication events, the diversification of paralogues and sequence constraint. They also confirm the significance of comparative proteomic and genomic studies that identified the molecular origins of synapses in unicellular eukaryotes and the vertebrate expansion in proteome complexity. These molecular mechanisms have general importance for understanding the repertoire of behaviours in different species and for human behavioural disorders arising from synapse gene mutations.     

Horizontal gene transfers as metagenomic gene duplications

While it is well accepted that horizontal gene transfer plays an important role in the evolution and the diversification of prokaryotic genomes, many questions remain open regarding its functional mechanisms of action and its interplay with the extant genome. This study addresses the relationship between proteome innovation by horizontal gene transfer and genome content in Proteobacteria. We characterize the transferred genes, focusing on the protein domain compositions and their relationships with the existing protein domain superfamilies in the genome. In agreement with previous observations, we find that the protein domain architectures of horizontally transferred genes are significantly shorter than the genomic average. Furthermore, protein domains that are more common in the total pool of genomes appear to have a proportionally higher chance to be transferred. This suggests that transfer events behave as if they were drawn randomly from a cross-genomic community gene pool, much like gene duplicates are drawn from a genomic gene pool. Finally, horizontally transferred genes carry domains of exogenous families less frequently for larger genomes, although they might do it more than expected by chance.     

Analysis of the compositional biases in Plasmodium falciparum genome and proteome using Arabidopsis thaliana as a reference

Comparative genomic analysis of the malaria causative agent, Plasmodium falciparum, with other eukaryotes for which the complete genome is available, revealed that the genome from P. falciparum was more similar to the genome of a plant, Arabidopsis thaliana, than to other non-apicomplexan taxa. Plant-like sequences are thought to result from horizontal gene transfers after a secondary endosymbiosis involving an algal ancestor. The use of the A. thaliana genome and proteome as a reference gives an opportunity to refine our understanding of the extreme compositional bias in the P. falciparum genome that leads to a proteome-wide amino acid bias. A set of pairs of non-redundant protein homologues was selected owing to rigorous genome-wide sequence comparison methods. The introduction of A. thaliana as a reference was a mean to weight the magnitude of the protein evolutionary divergence in P. falciparum. The correlation of the amino acid proportions with evolutionary time supports the hypothesis that amino acids encoded by GC-rich codons are directionally substituted into amino acids encoded by AT-rich codons in the P. falciparum proteome. The long-term deviation of codons in malarial sequences appears as a possible consequence of a genome-wide tri-nucleotidic signature imprinting. Additionally, this study suggests possible working guidelines to improve the accuracy of P. falciparum sequence comparisons, for homology searches and phylogenetic studies.     

Genomic sequencing reveals historical,demographic and selective factors associated with the diversification of the fire‐associated fungus Neurospora discreta

Delineating microbial populations, discovering ecologically relevant phenotypes and identifying migrants, hybrids or admixed individuals have long proved notoriously difficult, thereby limiting our understanding of the evolutionary forces at play during the diversification of microbial species. However, recent advances in sequencing and computational methods have enabled an unbiased approach whereby incipient species and the genetic correlates of speciation can be identified by examining patterns of genomic variation within and between lineages. We present here a population genomic study of a phylogenetic species in the Neurospora discreta species complex, based on the resequencing of full genomes (~37 Mb) for 52 fungal isolates from nine sites in three continents. Population structure analyses revealed two distinct lineages in South–East Asia, and three lineages in North America/Europe with a broad longitudinal and latitudinal range and limited admixture between lineages. Genome scans for selective sweeps and comparisons of the genomic landscapes of diversity and recombination provided no support for a role of selection at linked sites on genomic heterogeneity in levels of divergence between lineages. However, demographic inference indicated that the observed genomic heterogeneity in divergence was generated by varying rates of gene flow between lineages following a period of isolation. Many putative cases of exchange of genetic material between phylogenetically divergent fungal lineages have been discovered, and our work highlights the quantitative importance of genetic exchanges between more closely related taxa to the evolution of fungal genomes. Our study also supports the role of allopatric isolation as a driver of diversification in saprobic microbes.     

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.     

Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides

A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.     

Imputation of genotypes from low- to high-density genotyping platforms and implications for genomic selection

The objective of this study was to quantify the accuracy achievable from imputing genotypes from a commercially available low-density marker panel (2730 single nucleotide polymorphisms (SNPs) following edits) to a commercially available higher density marker panel (51 602 SNPs following edits) in Holstein-Friesian cattle using Beagle, a freely available software package. A population of 764 Holstein-Friesian animals born since 2006 were used as the test group to quantify the accuracy of imputation, all of which had genotypes for the high-density panel; only SNPs on the low-density panel were retained with the remaining SNPs to be imputed. The reference population for imputation consisted of 4732 animals born before 2006 also with genotypes on the higher density marker panel. The concordance between the actual and imputed genotypes in the test group of animals did not vary across chromosomes and was on average 95%; the concordance between actual and imputed alleles was, on average, 97% across all SNPs. Genomic predictions were undertaken across a range of production and functional traits for the 764 test group animals using either their real or imputed genotypes. Little or no mean difference in the genomic predictions was evident when comparing direct genomic values (DGVs) using real or imputed genotypes. The average correlation between the DGVs estimated using the real or imputed genotypes for the 15 traits included in the Irish total merit index was 0.97 (range of 0.92 to 0.99), indicating good concordance between proofs from real or imputed genotypes. Results show that a commercially available high-density marker panel can be imputed from a commercially available lower density marker panel, which will also have a lower cost, thereby facilitating a reduction in the cost of genomic selection. Increased available numbers of genotyped and phenotyped animals also has implications for increasing the accuracy of genomic prediction in the entire population and thus genetic gain using genomic selection.     

Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering.

Plaque-to-plaque transfers of RNA viruses lead to accumulation of mutations and fitness decrease. To test whether continuing plaque-to-plaque transfers would lead to viral extinction, we have subjected several low fitness foot-and-mouth disease virus (FMDV) clones to up to 130 successive plaque transfers, and have analyzed the evolution of plaque titers and genomic nucleotide sequences. No case of viral extinction could be documented. Some low fitness clones that posses an internal poly(A) tract evaded extinction by modifying the length or base composition of the poly(A) tract. The comparison of entire genomic sequences of FMDV clones at increasing plaque transfer number revealed that mutations accumulated at a uniform rate, and that they were distributed unevenly along the genome. Clusters of mutations were identified at different genomic sites in two plaque transfer lineages. Mutation clustering appears to occur stochastically and could not be related to fixation of compensatory mutations. The results document resistance of viral clones to extinction, and suggest that mutation clustering may be a mechanism of genetic diversification of low fitness virus.     

Comparative proteomics reveals evidence for evolutionary diversification of rodent seminal fluid and its functional significance in sperm competition

During insemination, males of internally fertilizing speciestransfer a complex array of seminal fluid proteins to the femalereproductive tract. These proteins can have profound effectson female reproductive physiology and behavior and are thoughtto mediate postcopulatory sexual selection and intersexual conflict.Such selection may cause seminal fluid to evolve rapidly, withpotentially important consequences for speciation. Here we investigatethe evolution of seminal fluid proteins in a major mammalianradiation, the muroid rodents, by quantifying diversity in seminalfluid proteome composition for the first time across a broadrange of closely related species. Using comparative proteomicstechniques to identify and cross-match proteins, we demonstratethat rodent seminal fluid is highly diverse at the level ofboth proteomes and individual proteins. The striking interspecificheterogeneity in seminal fluid composition revealed by our surveyfar exceeds that seen in a second proteome of comparable complexity,skeletal muscle, indicating that the complement of proteinsexpressed in seminal fluid may be subject to rapid diversification.We further show that orthologous seminal fluid proteins exhibitsubstantial interspecific variation in molecular mass. Becausethis variation cannot be attributed to differential glycosylationor radical differences in termination sites, it is stronglysuggestive of rapid amino acid divergence. Sperm competitionis implicated in generating such divergence for at least onemajor seminal fluid protein in our study, SVS II, which is responsiblefor copulatory plug formation via transglutaminase-catalyzedcross-linking after insemination. We show that the molecularmass of SVS II is positively correlated with relative testissize across species, which could be explained by selection foran increased number of cross-linking sites involved in the formationof the copulatory plug under sperm competition.     

An Indexed,Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.     

Recent advances in proteomic profiling of human blood: clinical scope

A chromosome-centric approach in combination with targeted selected reaction monitoring-mass spectrometry analysis is one of the main approaches to study the human proteome. Measuring the size of the human plasma proteome includes both definition of all forms of proteins and quantitative measuring of the content of each protein form. The algorithm for measuring the proteome of canonical (master) proteins of chromosome 18 was created by combining a chromosome-centric approach and selected reaction monitoring-mass spectrometry. It can be scaled for all chromosomes to measure master proteins in the human blood plasma. Establishment of selected reaction monitoring-mass spectrometry diagnostic assays for quantitative measuring of the proteins associated with the development of diseases is a practical result.     

A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome

The wealth of genomic data available for many organisms has set the stage for the next phase of structure-function analysis. High-throughput structural genomics is currently the method of choice for rapid analysis of protein structure-function relationships on a proteome-wide basis. The Joint Center for Structural Genomics (JCSG), established in 2000 under the NIH/NIGMS Protein Structure Initiative, has developed and implemented an integrated high-throughput structure pipeline and applied it in a 2-tiered approach to mining the proteome of the thermophilic bacterium Thermotoga maritima. In the first tier, the successful application of this integrated pipeline has resulted in the cloning and expression of 73% of the T. maritima proteome (1376 out of 1877 predicted genes), and has identified 465 proteins which produced crystal hits. These 465 proteins were compared with existing structural information and a subset of 269 targets were selected to process towards structure determination in a second tier effort. To date, the JCSG pipeline applied to the Thermotoga maritima proteome has resulted in 55 new structures and has identified 6 novel folds and continues to identify structures with novel features.     

The Proteome Analysis database: a tool for the in silico analysis of whole proteomes

The Proteome Analysis database (http://www.ebi.ac.uk/proteome/) has been developed by the Sequence Database Group at EBI utilizing existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archeae and eukaryotes. Three main projects are used, InterPro, CluSTr and GO Slim, to give an overview on families, domains, sites, and functions of the proteins from each of the complete genomes. Complete proteome analysis is available for a total of 89 proteome sets. A specifically designed application enables InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.     

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.