Non-redundant Protein Database Research Articles

EST analysis on the gonad development related organs and microarray screen for differentially expressed genes in mature ovary and testis of Scylla paramamosain

A total of 5160 high quality ESTs (expressed sequence tags) averaging 357 bp were collected from normalized cDNA libraries created from testis, ovary and mixed organs of mud crab Scylla paramamosain. Clustering and assembly of these ESTs resulted in a total of 3837 unique sequences with 576 overlapping contigs and 3261 singletons. Comparisons with the GenBank non-redundant (Nr) protein database (BLASTx, e-values < 10 − 5 ) revealed putative functions or matched homologs from other organisms for 847 (22%) of the ESTs. Several gonad development related genes such as cathepsin C, thioredoxin peroxidase, vitellogenin receptor precursor, 50S ribosomal protein L24 and ubiquitin-conjugating enzyme E2 isoform 2 were identified from this EST project and demonstrated as gonad differential expression genes by rqRT-PCR. Sixty five different types of SSRs (simple sequence repeats) were identified from the total 411 EST-SSR motifs. A home-made cDNA microarray containing 5664 spots was developed and the hybridization results indicated that 39 unique transcripts were differentially expressed in testis and ovaries (P < 0.05). The expression levels of eleven unique transcripts examined by rqRT-PCR were matched with microarray fairly. These results will provide a useful resource for functional genomic studies on the biology of reproduction of mud crab.

Uncovering the Human Methyltransferasome

We present a comprehensive analysis of the human methyltransferasome. Primary sequences, predicted secondary structures, and solved crystal structures of known methyltransferases were analyzed by hidden Markov models, Fisher-based statistical matrices, and fold recognition prediction-based threading algorithms to create a model, or profile, of each methyltransferase superfamily. These profiles were used to scan the human proteome database and detect novel methyltransferases. 208 proteins in the human genome are now identified as known or putative methyltransferases, including 38 proteins that were not annotated previously. To date, 30% of these proteins have been linked to disease states. Possible substrates of methylation for all of the SET domain and SPOUT methyltransferases as well as 100 of the 131 seven-β-strand methyltransferases were surmised from sequence similarity clusters based on alignments of the substrate-specific domains.

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.

A transcriptome analysis of mitten crab testes (Eriocheir sinensis)

The identification of expressed genes involved in sexual precocity of the mitten crab (Eriocheir sinensis) is critical for a better understanding of its reproductive development. To this end, we constructed a cDNA library from the rapid developmental stage of testis of E. sinensis and sequenced 3,388 randomly picked clones. After processing, 2,990 high-quality expressed sequence tags (ESTs) were clustered into 2,415 unigenes including 307 contigs and 2,108 singlets, which were then compared to the NCBI non-redundant (nr) protein and nucleotide (nt) database for annotation with Blastx and Blastn, respectively. After further analysis, 922 unigenes were obtained with concrete annotations and 30 unigenes were found to have functions possibly related to the process of reproduction in male crabs – six transcripts relevant to spermatogenesis (especially Cyclin K and RecA homolog DMC1), two transcripts involved in nuclear protein transformation, two heat-shock protein genes, eleven transcription factor genes (a series of zinc-finger proteins), and nine cytoskeleton protein-related genes. Our results, besides providing valuable information related to crustacean reproduction, can also serve as a base for future studies of reproductive and developmental biology.

Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation

Xylella fastidiosa ( Xf) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf-inoculation. We comparatively analyzed proteins differentially expressed in Xf-inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica. Total proteins were extracted from the stems of uninoculated controls and Xf-inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf-inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf, and heat shock protein 21 was not detectable in the Xf-inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf-inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf-inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.

High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

BackgroundBathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology.ResultsA normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR) and their RNA transcription level by quantitative PCR (qPCR) experiments.ConclusionsWe have established the first tissue transcriptional analysis of a deep-sea hydrothermal vent animal and generated a searchable catalog of genes that provides a direct method of identifying and retrieving vast numbers of novel coding sequences which can be applied in gene expression profiling experiments from a non-conventional model organism. This provides the most comprehensive sequence resource for identifying novel genes currently available for a deep-sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels.The characterization of the B. azoricus transcriptome will facilitate research into biological processes underlying physiological adaptations to hydrothermal vent environments and will provide a basis for expanding our understanding of genes putatively involved in adaptations processes during post-capture long term acclimatization experiments, at "sea-level" conditions, using B. azoricus as a model organism.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Assembly of the 221,746 sequence reads resulted in 12,442 contigs. Over 50 % (6183) of 12,442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

Domain-Based Identification and Analysis of Glutamate Receptor Ion Channels and Their Relatives in Prokaryotes

Voltage-gated and ligand-gated ion channels are used in eukaryotic organisms for the purpose of electrochemical signaling. There are prokaryotic homologues to major eukaryotic channels of these sorts, including voltage-gated sodium, potassium, and calcium channels, Ach-receptor and glutamate-receptor channels. The prokaryotic homologues have been less well characterized functionally than their eukaryotic counterparts. In this study we identify likely prokaryotic functional counterparts of eukaryotic glutamate receptor channels by comprehensive analysis of the prokaryotic sequences in the context of known functional domains present in the eukaryotic members of this family. In particular, we searched the nonredundant protein database for all proteins containing the following motif: the two sections of the extracellular glutamate binding domain flanking two transmembrane helices. We discovered 100 prokaryotic sequences containing this motif, with a wide variety of functional annotations. Two groups within this family have the same topology as eukaryotic glutamate receptor channels. Group 1 has a potassium-like selectivity filter. Group 2 is most closely related to eukaryotic glutamate receptor channels. We present analysis of the functional domain architecture for the group of 100, a putative phylogenetic tree, comparison of the protein phylogeny with the corresponding species phylogeny, consideration of the distribution of these proteins among classes of prokaryotes, and orthologous relationships between prokaryotic and human glutamate receptor channels. We introduce a construct called the Evolutionary Domain Network, which represents a putative pathway of domain rearrangements underlying the domain composition of present channels. We believe that scientists interested in ion channels in general, and ligand-gated ion channels in particular, will be interested in this work. The work should also be of interest to bioinformatics researchers who are interested in the use of functional domain-based analysis in evolutionary and functional discovery.

Biochemical Characterization of the Cell-Biomaterial Interface by Quantitative Proteomics

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.

Extended latex proteome analysis deciphers additional roles of the lettuce laticifer

Lettuce is an economically important leafy vegetable that accumulates a milk-like sap called latex in the laticifer. Previously, we conducted a large-scale lettuce latex proteomic analysis. However, the identified proteins were obtained only from lettuce ESTs and proteins deposited in NCBI databases. To extend the number of known latex proteins, we carried out an analysis identifying 302 additional proteins that were matched to the NCBI non-redundant protein database. Interestingly, the newly identified proteins were not recovered from lettuce EST and protein databases, indicating the usefulness of this hetero system in MudPIT analysis. Gene ontology studies revealed that the newly identified latex proteins are involved in many processes, including many metabolic pathways, binding functions, stress responses, developmental processes, protein metabolism, transport and signal transduction. Application of the non-redundant plant protein database led to the identification of an increased number of latex proteins. These newly identified latex proteins provide a rich source of information for laticifer research.

Proteomic analysis of germinating urediniospores of Phakopsora pachyrhizi , causal agent of Asian soybean rust

Phakopsora pachyrhizi is an obligate pathogen that causes Asian soybean rust. Asian soybean rust has an unusually broad host range and infects by direct penetration through the leaf cuticle. In order to understand the early events in the infection process, it is important to identify and characterize proteins in P. pachyrhizi. Germination of the urediniospore is the first stage in the infection process and represents a critical life stage applicable to studies with this obligate pathogen. We have applied a 2-DE and MS approach to identify 117 proteins from the National Center of Biotechnology Information nonredundant protein database and a custom database of Basidiomycota EST sequences. Proteins with roles in primary metabolism, energy transduction, stress, cellular regulation and signaling were identified in this study. This data set is accessible at http://world-2dpage.expasy.org/repository/database=0018.

Integrated Phosphoproteomics Analysis of a Signaling Network Governing Nutrient Response and Peroxisome Induction

Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.

Generation and analysis of expressed sequence tags from adductor muscle of Japanese scallop Mizuhopecten yessoensis

A normalized cDNA library was constructed from the adductor muscle of M. yessoensis and acquired 4595 high quality expressed sequence tags (ESTs). After clustering and assembly of the ESTs, 3061 unigenes containing 654 contigs and 2407 singletons were identified. The contig length ranged from 266 bp to 2364 bp and the average length of these contigs was 544 bp. Blastx nonredundant protein database analysis showed that 1522 unigenes had significant homology to known genes (E value ≤ 10 − 5 ). By comparing to Clusters of Orthologous Groups (COG) categories, 460 unigenes were annotated (E value ≤ 10 − 10 ). Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 345 of 3061 unigenes were assigned into 103 pathways (E value ≤ 10 − 5 ). For InterProScan searches, 1237 unigenes were annotated containing 727 different types of protein domains. 941 of the 1237 unigenes were annotated for Gene Ontology (GO) classification using Uniprot2GO associations in any category (biological, cellular, and molecular). By sequences comparability and analysis of Blastx NCBI nonredundant protein database and KEGG, 66 unigenes were identified that may be involved in genetic information processing based on the known knowledge. The study provides a material basis as useful information for the genomic analysis of shellfish.

Requirement of a Homolog of Glucosidase II β-Subunit for EFR-Mediated Defense Signaling in Arabidopsis thaliana

EFR is a plasma-membrane resident receptor responsible for recognition of microbial elongation factor Tu (EF-Tu) and thus triggering plant innate immunity to fend off phytopathogens. Functional EFR must be subject to the endoplasmic reticulum quality control (ERQC) machinery for the correct folding and proper assembly in order to reach its final destination. Genetic studies have demonstrated that ERD2b, a counterpart of the yeast or mammalian HDEL receptor ERD2 for retaining proteins in the endoplasmic reticulum (ER) lumen, is required for EFR function in plants (Li et al., 2009). In this study, we characterized the Arabidopsis glucosidase II beta-subunit via the HDEL motif against the non-redundant protein database. Data mining also revealed that the glucosidase II beta-subunit gene has a highly similar expression pattern to ERD2b and the other known ERQC components involved in EFR biogenesis. Importantly, the T-DNA insertion lines of the glucosidase II beta-subunit gene showed that EFR-controlled responses were substantially reduced or completely blocked in these mutants. The responses include seedling growth inhibition, induction of marker genes, MAP kinase activation, and callose deposition, triggered by peptide elf18, a full mimic of EF-Tu. Taken together, our data indicate a requirement of the glucosidase II beta-subunit for EFR function.

Analysis of expressed sequence tags for Frankliniella occidentalis, the western flower thrips

Thrips are members of the insect order Thysanoptera and Frankliniella occidentalis (the western flower thrips) is the most economically important pest within this order. F. occidentalis is both a direct pest of crops and an efficient vector of plant viruses, including Tomato spotted wilt virus (TSWV). Despite the world-wide importance of thrips in agriculture, there is little knowledge of the F. occidentalis genome or gene functions at this time. A normalized cDNA library was constructed from first instar thrips and 13 839 expressed sequence tags (ESTs) were obtained. Our EST data assembled into 894 contigs and 11 806 singletons (12 700 nonredundant sequences). We found that 31% of these sequences had significant similarity (E< or = 10(-10)) to protein sequences in the National Center for Biotechnology Information nonredundant (nr) protein database, and 25% were functionally annotated using Blast 2GO. We identified 74 sequences with putative homology to proteins associated with insect innate immunity. Sixteen sequences had significant similarity to proteins associated with small RNA-mediated gene silencing pathways (RNA interference; RNAi), including the antiviral pathway (short interfering RNA-mediated pathway). Our EST collection provides new sequence resources for characterizing gene functions in F. occidentalis and other thrips species with regards to vital biological processes, studying the mechanism of interactions with the viruses harboured and transmitted by the vector, and identifying new insect gene-centred targets for plant disease and insect control.

Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities ( E value < 10 − 5 ) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification.

Evolution and function of the plant cell wall synthesis-related glycosyltransferase family 8.

Carbohydrate-active enzyme glycosyltransferase family 8 (GT8) includes the plant galacturonosyltransferase1-related gene family of proven and putative alpha-galacturonosyltransferase (GAUT) and GAUT-like (GATL) genes. We computationally identified and investigated this family in 15 fully sequenced plant and green algal genomes and in the National Center for Biotechnology Information nonredundant protein database to determine the phylogenetic relatedness of the GAUTs and GATLs to other GT8 family members. The GT8 proteins fall into three well-delineated major classes. In addition to GAUTs and GATLs, known or predicted to be involved in plant cell wall biosynthesis, class I also includes a lower plant-specific GAUT and GATL-related (GATR) subfamily, two metazoan subfamilies, and proteins from other eukaryotes and cyanobacteria. Class II includes galactinol synthases and plant glycogenin-like starch initiation proteins that are not known to be directly involved in cell wall synthesis, as well as proteins from fungi, metazoans, viruses, and bacteria. Class III consists almost entirely of bacterial proteins that are lipooligo/polysaccharide alpha-galactosyltransferases and alpha-glucosyltransferases. Sequence motifs conserved across all GT8 subfamilies and those specific to plant cell wall-related GT8 subfamilies were identified and mapped onto a predicted GAUT1 protein structure. The tertiary structure prediction identified sequence motifs likely to represent key amino acids involved in catalysis, substrate binding, protein-protein interactions, and structural elements required for GAUT1 function. The results show that the GAUTs, GATLs, and GATRs have a different evolutionary origin than other plant GT8 genes, were likely acquired from an ancient cyanobacterium (Synechococcus) progenitor, and separate into unique subclades that may indicate functional specialization.

A Comprehensive Analysis of Structural and Sequence Conservation in the TetR Family Transcriptional Regulators

The tetracycline repressor family transcriptional regulators (TFRs) are homodimeric DNA-binding proteins that generally act as transcriptional repressors. Their DNA-binding activity is allosterically inactivated by the binding of small-molecule ligands. TFRs constitute the third most frequently occurring transcriptional regulator family found in bacteria with more than 10,000 representatives in the nonredundant protein database. In addition, more than 100 unique TFR structures have been solved by X-ray crystallography. In this study, we have used computational and experimental approaches to reveal the variations and conservation present within TFRs. Although TFR structures are very diverse, we were able to identify a conserved central triangle in their ligand-binding domains that forms the foundation of the structure and the framework for the ligand-binding cavity. While the sequences of DNA-binding domains of TFRs are highly conserved across the whole family, the sequences of their ligand-binding domains are so diverse that pairwise sequence similarity is often undetectable. Nevertheless, by analyzing subfamilies of TFRs, we were able to identify distinct regions of conservation in ligand-binding domains that may be important for allostery. To aid in large-scale analyses of TFR function, we have developed a simple and reliable computational approach to predict TFR operator sequences, a temperature melt-based assay to measure DNA binding, and a generic ligand-binding assay that will likely be applicable to most TFRs. Finally, our analysis of TFR structures highlights their flexibility and provides insight into a conserved allosteric mechanism for this family.

Improving Hox Protein Classification across the Major Model Organisms

The family of Hox-proteins has been a major focus of research for over 30 years. Hox-proteins are crucial to the correct development of bilateral organisms, however, some uncertainty remains as to which Hox-proteins are functionally equivalent across different species. Initial classification of Hox-proteins was based on phylogenetic analysis of the 60 amino acid homeodomain. This approach was successful in classifying Hox-proteins with differing homeodomains, but the relationships of Hox-proteins with nearly identical homeodomains, yet distinct biological functions, could not be resolved. Correspondingly, these ‘problematic’ proteins were classified into one large unresolved group. Other classifications used the relative location of the Hox-protein coding genes on the chromosome (synteny) to further resolve this group. Although widely used, this synteny-based classification is inconsistent with experimental evidence from functional equivalence studies. These inconsistencies led us to re-examine and derive a new classification for the Hox-protein family using all Hox-protein sequences available in the GenBank non-redundant protein database (NCBI-nr). We compare the use of the homeodomain, the homeodomain with conserved flanking regions (the YPWM and linker region), and full length Hox-protein sequences as a basis for classification of Hox-proteins. In contrast to previous attempts, our approach is able to resolve the relationships for the ‘problematic’ as well as ABD-B-like Hox-proteins. We highlight differences to previous classifications and clarify the relationships of Hox-proteins across the five major model organisms, Caenorhabditis elegans, Drosophila melanogaster, Branchiostoma floridae, Mus musculus and Danio rerio. Comparative and functional analysis of Hox-proteins, two fields crucial to understanding the development of bilateral organisms, have been hampered by difficulties in predicting functionally equivalent Hox-proteins across species. Our classification scheme offers a higher-resolution classification that is in accordance with phylogenetic as well as experimental data and, thereby, provides a novel basis for experiments, such as comparative and functional analyses of Hox-proteins.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes

BackgroundSugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at ~12×. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level.ResultsThe microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons.ConclusionsThe sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.