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Results for “Molecular Sequence Annotation”

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PubMed2003

Computational detection of cis -regulatory modules.

MOTIVATION: The transcriptional regulation of a metazoan gene depends on the cooperative action of multiple transcription factors that bind to cis-regulatory modules (CRMs) located in the neighborhood of the gene. By integrating multiple signals, CRMs confer an organism specific spatial and temporal rate of transcription. RESULTS: Based on the hypothesis that genes that are needed in exactly the same conditions might share similar regulatory switches, we have developed a novel methodology to find CRMs in a set of coexpressed or coregulated genes. The ModuleSearcher algorithm finds for a given gene set the best scoring combination of transcription factor binding sites within a sequence window using an A(*)procedure for tree searching. To keep the level of noise low, we use DNA sequences that are most likely to contain functional cis-regulatory information, namely conserved regions between human and mouse orthologous genes. The ModuleScanner performs genomic searches with a predicted CRM or with a user-defined CRM known from the literature to find possible target genes. The validity of a set of putative targets is checked using Gene Ontology annotations. We demonstrate the use and effectiveness of the ModuleSearcher and ModuleScanner algorithms and test their specificity and sensitivity on semi-artificial data. Next, we search for a module in a cluster of gene expression profiles of human cell cycle genes. AVAILABILITY: The ModuleSearcher is available as a web service within the TOUCAN workbench for regulatory sequence analysis, which can be downloaded from http://www.esat.kuleuven.ac.be/~dna/BioI.

Algorithms↗

PubMed2001

Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis.

A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

Alleles↗

PubMed2003

Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome.

Increasingly complex bioinformatic analysis is necessitated by the plethora of sequence information currently available. A total of 21 poxvirus genomes have now been completely sequenced and annotated, and many more genomes will be available in the next few years. First, we describe the creation of a database of continuously corrected and updated genome sequences and an easy-to-use and extremely powerful suite of software tools for the analysis of genomes, genes, and proteins. These tools are available free to all researchers and, in most cases, alleviate the need for using multiple Internet sites for analysis. Further, we describe the use of these programs to identify conserved families of genes (poxvirus orthologous clusters) and have named the software suite POCs, which is available at www.poxvirus.org. Using POCs, we have identified a set of 49 absolutely conserved gene families-those which are conserved between the highly diverged families of insect-infecting entomopoxviruses and vertebrate-infecting chordopoxviruses. An additional set of 41 gene families conserved in chordopoxviruses was also identified. Thus, 90 genes are completely conserved in chordopoxviruses and comprise the minimum essential genome, and these will make excellent drug, antibody, vaccine, and detection targets. Finally, we describe the use of these tools to identify necessary annotation and sequencing updates in poxvirus genomes. For example, using POCs, we identified 19 genes that were widely conserved in poxviruses but missing from the vaccinia virus strain Tian Tan 1998 GenBank file. We have reannotated and resequenced fragments of this genome and verified that these genes are conserved in Tian Tan. The results for poxvirus genes and genomes are discussed in light of evolutionary processes.

Amino Acid Sequence↗

PubMed2006

Choosing negative examples for the prediction of protein-protein interactions.

The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions.

Algorithms↗

PubMed2005

Retroviral insertional mutagenesis: past, present and future.

Retroviral insertion mutagenesis screens in mice are powerful tools for efficient identification of oncogenic mutations in an in vivo setting. Many oncogenes identified in these screens have also been shown to play a causal role in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly facilitated identification of oncogenic events in retrovirus-induced tumours. In this review, we discuss the features of retroviral insertion mutagenesis screens, covering the mechanisms by which retroviral insertions mutate cellular genes, the practical aspects of insertion site cloning, the identification and analysis of common insertion sites, and finally we address the potential for use of somatic insertional mutagens in the study of nonhaematopoietic and nonmammary tumour types.

Cloning, Molecular↗

PubMed2005

Elicitin genes in Phytophthora infestans are clustered and interspersed with various transposon-like elements.

Sequencing and annotation of a contiguous stretch of genomic DNA (112.3 kb) from the oomycete plant pathogen Phytophthora infestans revealed the order, spacing and genomic context of four members of the elicitin (inf) gene family. Analysis of the GC content at the third codon position (GC3) of six genes encoded in the region, and a set of randomly selected coding regions as well as random genomic regions, showed that a high GC3 value is a general feature of Phytophthora genes that can be exploited to optimize gene prediction programs for Phytophthora species. At least one-third of the annotated 112.3-kb P. infestans sequence consisted of transposons or transposon-like elements. The most prominent were four Tc3/gypsy and Tc1/copia type retrotransposons and three DNA transposons that belong to the Tc1/mariner, Pogo and PiggyBac groups, respectively. Comparative analysis of other available genomic sequences suggests that transposable elements are highly heterogeneous and ubiquitous in the P. infestans genome.

Algal Proteins↗

PubMed2003

Consistency checks for characterizing protein forms.

Proteomics enforces the reverse chronological order on the gene to protein dogma and imposes amino acid sequences as a starting point of an investigation relative to function. By this approach, proteomics data can confirm the presence of multiple forms of a protein. Notwithstanding variations attributed specific individual features of organisms and tissues, from two to over ten protein forms can be identified in a given sample. The present work describes some guidelines for tracking the origin of alternative protein forms and attempts to tag the details of sequence data in the literature. Working via these guidelines we have uncovered a third alternative form of the Pim subfamily of oncogenes. The term form is here combined with the qualification alternative to describe any product of a given gene including closely related paralogs. This paper also emphasizes the need for consistency checks in annotation processes, such as gene clustering, to avoid losing important details describing protein alternative forms. By identifying alternative protein forms, we illustrate the fact that rationalizing of protein function via the identification of protein-protein interactions should in reality be that of identifying (alternative) form-form interactions.

Amino Acid Sequence↗

PubMed2026

Integrated Genome Mining and Bioactivity-Guided Isolation of Antimicrobial Peptides from Bacillus amyloliquefaciens BS4.

Bacterial resistance remains a critical global health challenge, driving the continuous search for novel antimicrobial agents. Bacillus amyloliquefaciens is a recognized repository of bioactive metabolites; however, its full biosynthetic potential requires integrated genomic and experimental validation. This study characterized the antimicrobial profile of B. amyloliquefaciens BS4 through a hybrid pipeline. Genome sequencing and de novo assembly revealed a 3.9 Mb chromosome with a G + C content of 46.14%. Functional annotation identified 3,887 coding sequences, including pathways for siderophore biosynthesis and a complete bacilysin biosynthetic cluster. BGC analysis using antiSMASH v7.1.0 and BAGEL4 identified 18 biosynthetic gene clusters, while similarity network analysis via BiG-SCAPE highlighted unique singleton BGCs, indicating untapped biosynthetic diversity. Although in silico screening via Macrel predicted two putative cationic antimicrobial peptides (AMPs), bioactivity-guided purification utilizing sequential RP-HPLC, and de novo sequencing revealed a distinct set of four active peptides. Notably, three of these sequences were identified as fragments derived from the BclA exosporium protein family, highlighting the structural proteome as a non-canonical source of antimicrobials. The purified fractions exhibited activity against M. luteus and E. coli, while displaying no significant hemolytic activity or cytotoxicity, even above the MIC values. Molecular docking further supported the interaction of these candidates with bacterial targets. Overall, this hybrid strategy effectively uncovers the antimicrobial complexity of BS4, revealing 'cryptic' peptide candidates with therapeutic potential.

Bacillus amyloliquefaciens BS4↗

PubMed2004

Functional annotation of the Arabidopsis genome using controlled vocabularies.

Controlled vocabularies are increasingly used by databases to describe genes and gene products because they facilitate identification of similar genes within an organism or among different organisms. One of The Arabidopsis Information Resource's goals is to associate all Arabidopsis genes with terms developed by the Gene Ontology Consortium that describe the molecular function, biological process, and subcellular location of a gene product. We have also developed terms describing Arabidopsis anatomy and developmental stages and use these to annotate published gene expression data. As of March 2004, we used computational and manual annotation methods to make 85,666 annotations representing 26,624 unique loci. We focus on associating genes to controlled vocabulary terms based on experimental data from the literature and use The Arabidopsis Information Resource-developed PubSearch software to facilitate this process. Each annotation is tagged with a combination of evidence codes, evidence descriptions, and references that provide a robust means to assess data quality. Annotation of all Arabidopsis genes will allow quantitative comparisons between sets of genes derived from sources such as microarray experiments. The Arabidopsis annotation data will also facilitate annotation of newly sequenced plant genomes by using sequence similarity to transfer annotations to homologous genes. In addition, complete and up-to-date annotations will make unknown genes easy to identify and target for experimentation. Here, we describe the process of Arabidopsis functional annotation using a variety of data sources and illustrate several ways in which this information can be accessed and used to infer knowledge about Arabidopsis and other plant species.

Arabidopsis↗

PubMed2002

N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence.

Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity. Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %).

Acyltransferases↗

PubMed2001

Identification of a cadmium-induced gene in Mycobacterium bovis and Mycobacterium tuberculosis.

A 17-kDa protein (CadI) was induced by cadmium in Mycobacterium bovis and Mycobacterium tuberculosis. Comparison of the N-terminal sequence from M. bovis CadI with the annotated M. tuberculosis genome database identified Rv2641 as the encoding gene. Long and short promoter fragments from M. bovis cadI were fused to the lacZ reporter gene in pYUB76. Only the long fragment directed cadmium-inducible activity when electroporated into M. bovis. The cadI promoter has potential for both constitutive and inducible expression studies in M. bovis and M. tuberculosis.

Amino Acid Sequence↗

PubMed2005

An extensible automated protein annotation tool: standardizing input and output using validated XML.

MOTIVATION: There is a frequent need to apply a large range of local or remote prediction and annotation tools to one or more sequences. We have created a tool able to dispatch one or more sequences to assorted services by defining a consistent XML format for data and annotations. RESULTS: By analyzing annotation tools, we have determined that annotations can be described using one or more of the six forms of data: numeric or textual annotation of residues, domains (residue ranges) or whole sequences. With this in mind, XML DTDs have been designed to store the input and output of any server. Plug-in wrappers to a number of services have been written which are called from a master script. The resulting APATML is then formatted for display in HTML. Alternatively further tools may be written to perform post-analysis.

Amino Acid Sequence↗

PubMed2002

Ab initio protein structure prediction.

Steady progress has been made in the field of ab initio protein folding. A variety of methods now allow the prediction of low-resolution structures of small proteins or protein fragments up to approximately 100 amino acid residues in length. Such low-resolution structures may be sufficient for the functional annotation of protein sequences on a genome-wide scale. Although no consistently reliable algorithm is currently available, the essential challenges to developing a general theory or approach to protein structure prediction are better understood. The energy landscapes resulting from the structure prediction algorithms are only partially funneled to the native state of the protein. This review focuses on two areas of recent advances in ab initio structure prediction-improvements in the energy functions and strategies to search the caldera region of the energy landscapes.

Chemistry, Physical↗

PubMed2005

Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay.

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.

Amino Sugars↗

PubMed1997

Isolation and regional mapping of cDNAs expressed during early human development.

A cDNA sequencing project was initiated with the aim of isolating and mapping new genes expressed during early human development. A human embryo cDNA library was constructed, and a prescreening procedure was used to select cDNAs corresponding to poorly transcribed genes. Partial sequences were generated from one or both ends of 231 cDNA clones, and sequence comparison with genetic databases revealed that 28% were already annotated genes, 42% matched with partial sequences expressed sequence tags that had already been detected, 3% contained no insert, 5% were highly similar to sequences from other species, and 23% of the cDNAs appeared to be unknown in genetic databases. All new sequences were deposited in public genetic databases, and most of the corresponding cDNAs were regionally mapped on human chromosome bands using both fluorescence and radioactive in situ hybridization. Several cDNAs colocalized with critical regions of the genome regarding mapped disorders, thus providing candidate genes for human genetic diseases.

Chromosome Mapping↗

PubMed2005

hATpin, a family of MITE-like hAT mobile elements conserved in diverse plant species that forms highly stable secondary structures.

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species.

Base Sequence↗

PubMed2005

Analysis of expressed sequence tags from Musa acuminata ssp. burmannicoides, var. Calcutta 4 (AA) leaves submitted to temperature stresses.

In order to discover genes expressed in leaves of Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA), from plants submitted to temperature stress, we produced and characterized two full-length enriched cDNA libraries. Total RNA from plants subjected to temperatures ranging from 5 degrees C to 25 degrees C and from 25 degrees C to 45 degrees C was used to produce a COLD and a HOT cDNA library, respectively. We sequenced 1,440 clones from each library. Following quality analysis and vector trimming, we assembled 2,286 sequences from both libraries into 1,019 putative transcripts, consisting of 217 clusters and 802 singletons, which we denoted Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Of these MaAES, 22.87% showed no matches with existing sequences in public databases. A global analysis of the MaAES data set indicated that 10% of the sequenced cDNAs are present in both cDNA libraries, while 42% and 48% are present only in the COLD or in the HOT libraries, respectively. Annotation of the MaAES data set categorized them into 22 functional classes. Of the 2,286 high-quality sequences, 715 (31.28%) originated from full-length cDNA clones and resulted in a set of 149 genes.

Base Sequence↗

PubMed2004

TcRRMs and Tcp28 genes are intercalated and differentially expressed in Trypanosoma cruzi life cycle.

The identification and characterization of RNA binding proteins in Trypanosoma cruzi are particularly relevant as they play key roles in the regulatory mechanisms of gene expression. In this work, we have identified coding sequences for the proteins, named TcRRM1 and TcRRM2, in the EST database generated by the T. cruzi genomic initiative. TcRRM1 and TcRRM2 contain two RNA binding domains (RRM) and are very similar to two Trypanosoma brucei RNA binding proteins previously reported, Tbp34 and Tbp37, and to a not yet annotated ORF in Leishmania major genome project. The T. cruzi RRM genes are organized in tandem, alternating with copies of Tcp28, a gene of unknown function. However, TcRRM transcript accumulation is higher in the spheromastigote stage, while Tcp28 transcripts accumulate more in the trypomastigote stage suggesting developmental regulation.

Amino Acid Sequence↗