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Results for “Molecular Sequence Annotation”

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PubMed2004

Prolinks: a database of protein functional linkages derived from coevolution.

The advent of whole-genome sequencing has led to methods that infer protein function and linkages. We have combined four such algorithms (phylogenetic profile, Rosetta Stone, gene neighbor and gene cluster) in a single database--Prolinks--that spans 83 organisms and includes 10 million high-confidence links. The Proteome Navigator tool allows users to browse predicted linkage networks interactively, providing accompanying annotation from public databases. The Prolinks database and the Proteome Navigator tool are available for use online at http://dip.doe-mbi.ucla.edu/pronav.

ATP Synthetase Complexes↗

PubMed1996

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

BRCA2 Protein↗

PubMed2004

Delineation of modular proteins: domain boundary prediction from sequence information.

The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of 'domain' do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.

Algorithms↗

PubMed2003

A comparative genomic analysis of the cow, pig, and human CFTR genes identifies potential intronic regulatory elements.

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.

Animals↗

PubMed1998

Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.

Diagnostic re-sequencing plays a central role in medical and evolutionary genetics. In this report we describe a process that applies fluorescence-based re-sequencing and an integrated set of analysis tools to automate and simplify the identification of DNA variations using the human mitochondrial genome as a model system. Two programs used in genome sequence analysis (Phred, a base-caller, and Phrap, a sequence assembler) are applied to assess the quality of each base call across the sequence. Potential DNA variants are automatically identified and 'tagged' by comparing the assembled sequence with a reference sequence. We also show that employing the Consed program to display a set of highly annotated reference sequences greatly simplifies data analysis by providing a visual database containing information on the location of the PCR primers, coding and regulatory sequences and previously known DNA variants. Among the 12 genomes sequenced 378 variants including 29 new variants were identified along with two heteroplasmic sites, automatically detected by the PolyPhred program. Overall we document the ease and speed of performing high quality and accurate fluorescence-based re-sequencing on long tracts of DNA as well as the application of new approaches to automatically find and view DNA variants among these sequences.

Base Sequence↗

PubMed2026

Identification of Rare Noncoding Variants in Familial Nonmedullary Thyroid Carcinoma.

BACKGROUND: Familial nonmedullary thyroid carcinoma (FNMTC) occurs when three or more family members are affected by usually papillary thyroid carcinoma (PTC), the most common form of NMTC. While the heritability to NMTC is among the highest of all cancers, the genetic determinants among NMTC families are not well understood. Here, we aim to understand the contribution of rare noncoding germline variants in the etiology of FNMTC. METHODS: We previously reported whole-genome sequencing (WGS) and linkage analysis in 17 PTC families and reported on 41 protein-coding variants in 40 genes that cosegregated with PTC in 11 of the families. Herein, we further leveraged our WGS data to include noncoding variants in our analysis for all 17 families. We hypothesized that most of the pathogenic noncoding variants would be located in theoretical or empirically determined regulatory regions that demonstrate at a minimum, basal thyroid expression, a positive family linkage score, and co-segregation among PTC-affected individuals. To test this hypothesis, we adopted a unique filtering strategy to identify variants that occurred in known DNA elements and transcription factor binding sites, near regions known to impact on gene expression or splicing in thyroid tissue, and/or in characterized thyroid enhancers. We annotated variants using two analyses (ENCODE and transcription factor binding site) within the BasePlayer software. We separately analyzed (1) expression quantitative trait loci, (2) splicing quantitative trait loci, and (3) thyroid enhancers. We then ranked variants according to predicted pathogenicity and performed Sanger sequencing in all individuals of each family. RESULTS: In total, 121 variants were selected based on in-silico prediction and our custom ranking analysis in each pedigree. Of these, 56 variants showed cosegregation among all PTC-affected individuals and were absent from unaffected individuals. This included candidate variants from five of the six PTC families for whom no protein-coding variants were previously found. CONCLUSION: Our data suggest that noncoding variants are important in the etiology of FNMTC and provide a framework for identifying noncoding germline variants using a novel approach. Further studies are needed to functionally characterize these variants to better understand the molecular mechanism of their pathogenicity.

Humans↗

PubMed2002

Inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking.

Higher plants produce several families of proteins with toxic properties, which act as defense compounds against pests and pathogens. The thionin family represents one family and comprises low molecular mass cysteine-rich proteins, usually basic and distributed in different plant tissues. Here, we report the purification and characterization of a new thionin from cowpea (Vigna unguiculata) with proteinase inhibitory activity. Cowpea thionin inhibits trypsin, but not chymotrypsin, binding with a stoichiometry of 1:1 as shown with the use of mass spectrometry. Previous annotations of thionins as proteinase inhibitors were based on their erroneous identification as homologues of Bowman-Birk family inhibitors. Molecular modeling experiments were used to propose a mode of docking of cowpea thionin with trypsin. Consideration of the dynamic properties of the cowpea thionin was essential to arrive at a model with favorable interface characteristics comparable with structures of trypsin-inhibitor complexes determined by X-ray crystallography. In the final model, Lys11 occupies the S1 specificity pocket of trypsin as part of a canonical style interaction.

Amino Acid Sequence↗

PubMed2006

Genomic and genetic characterization of rice Cen3 reveals extensive transcription and evolutionary implications of a complex centromere.

The centromere is the chromosomal site for assembly of the kinetochore where spindle fibers attach during cell division. In most multicellular eukaryotes, centromeres are composed of long tracts of satellite repeats that are recalcitrant to sequencing and fine-scale genetic mapping. Here, we report the genomic and genetic characterization of the complete centromere of rice (Oryza sativa) chromosome 3. Using a DNA fiber-fluorescence in situ hybridization approach, we demonstrated that the centromere of chromosome 3 (Cen3) contains approximately 441 kb of the centromeric satellite repeat CentO. Cen3 includes an approximately 1,881-kb domain associated with the centromeric histone CENH3. This CENH3-associated chromatin domain is embedded within a 3,113-kb region that lacks genetic recombination. Extensive transcription was detected within the CENH3 binding domain based on comprehensive annotation of protein-coding genes coupled with empirical measurements of mRNA levels using RT-PCR and massively parallel signature sequencing. Genes <10 kb from the CentO satellite array were expressed in several rice tissues and displayed histone modification patterns consistent with euchromatin, suggesting that rice centromeric chromatin accommodates normal gene expression. These results support the hypothesis that centromeres can evolve from gene-containing genomic regions.

Centromere↗

PubMed2003

Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels.

BACKGROUND: The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions. RESULTS: We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity. CONCLUSIONS: Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.

Alternative Splicing↗

PubMed1998

HOMSTRAD: a database of protein structure alignments for homologous families.

We describe a database of protein structure alignments for homologous families. The database HOMSTRAD presently contains 130 protein families and 590 aligned structures, which have been selected on the basis of quality of the X-ray analysis and accuracy of the structure. For each family, the database provides a structure-based alignment derived using COMPARER and annotated with JOY in a special format that represents the local structural environment of each amino acid residue. HOMSTRAD also provides a set of superposed atomic coordinates obtained using MNYFIT, which can be viewed with a graphical user interface or used for comparative modeling studies. The database is freely available on the World Wide Web at: http://www-cryst.bioc.cam. ac.uk/-homstrad/, with search facilities and links to other databases.

Aspartic Acid Endopeptidases↗

PubMed2006

Functional characterization of ketoreductase (rubN6) and aminotransferase (rubN4) genes in the gene cluster of Streptomyces achromogenes var. rubradiris.

ORF's for rubN6 and rubN4 have been annotated as thymidine diphosphate glucose 4-ketoreductase and thymidine diphosphate glucose 3-aminotransferase by sequence analysis of the rubradirin biosynthetic gene cluster cloned from Streptomyces achromogenes var. rubradiris NRRL 3061. Both ORFs were heterologously expressed in Escherichia coli as His-tagged fusion proteins. The functionalities of TDP-glucose 4-ketoreductase and TDP-glucose 3-aminotransferase were verified by in vitro enzyme assay, and a biosynthetic pathway for TDP-D: -rubranitrose is proposed.

Amino Acid Sequence↗

PubMed2004

The Mycobacterium tuberculosis Rv1099c gene encodes a GlpX-like class II fructose 1,6-bisphosphatase.

There are now abundant data indicating that Mycobacterium tuberculosis uses fatty acids as a carbon source in vivo. A key enzyme in gluconeogenesis, missing in the original annotation of the M. tuberculosis genome, is fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11). The authors have shown that M. tuberculosis Rv1099c, a glpX homologue, can complement Escherichia coli mutants lacking FBPase. The protein encoded by Rv1099c was shown to have FBPase activity. Rv1099c was expressed at significant levels in M. tuberculosis, and may encode the major FBPase of this pathogen.

Escherichia coli↗

PubMed2000

A novel type of RNase III family proteins in eukaryotes.

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. We isolated the drosha gene, a new member of this family in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila drosha throughout its entire protein sequence. A potential drosha homologue is also found among the deposited human cDNA sequences. A comparison of these drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family.

Amino Acid Sequence↗

PubMed2000

PAHdb: a locus-specific knowledgebase.

PAHdb is an online relational locus-specific "mutation database" (http://www.mcgill.ca/pahdb) for the human phenylalanine hydroxylase gene (symbol PAH) and its associated phenotypes (protein, metabolic, clinical). When combined with associated information (population distribution of allele, haplotype association, etc.) PAHdb functions as a knowledgebase. From the outset, and in the absence of raw data (e.g., sequence gels), PAHdb has instead been an annotated repository of information about mutations maintained by a team of curators. It is also disease-oriented, being focused on a variant phenotype (hyperphenylalaninemia (HPA) and its most important form of disease, phenylketonuria (PKU)) resulting from primary dysfunction of the PAH enzyme (EC 1.14.16.1); it is "patient friendly" in that it contains information for those personally involved with HPA/PKU (MIM# 261600). PAHdb also serves its community through direct interaction.

Alleles↗

PubMed2005

Hemiascomycetous yeasts at the forefront of comparative genomics.

With more than a dozen species fully sequenced, as many as this partially sequenced, and more in development, yeasts are now used to explore the frontlines of comparative genomics of eukaryotes. Innovative procedures have been developed to compare and annotate genomes at various evolutionary distances, to identify short cis-acting regulatory elements, to map duplications, or to align syntenic blocks. Human and plant pathogens, in addition to yeasts that show a variety of interesting physiological properties, are included in this multidimensional comparative survey, which encompasses a very broad evolutionary range. As major steps of the evolutionary history of hemiascomycetous genomes emerge, precise questions on the general mechanisms of their evolution can be addressed, using both experimental and in silico methods.

Adaptation, Physiological↗

PubMed2005

Discovering hidden viral piracy.

MOTIVATION: Viruses and developers of anti-inflammatory therapies share a common interest in proteins that manipulate the immune response. Large double-stranded DNA viruses acquire host proteins to evade host defense mechanisms. Hence, viral pirated proteins may have a therapeutic potential. Although dozens of viral piracy events have already been identified, we hypothesized that sequence divergence impedes the discovery of many others. RESULTS: We developed a method to assess the number of viral/human homologs and discovered that at least 917 highly diverged homologs are hidden in low-similarity alignment hits that are usually ignored. However, these low-similarity homologs are masked by many false alignment hits. We therefore applied a filtering method to increase the proportion of viral/human homologous proteins. The homologous proteins we found may facilitate functional annotation of viral and human proteins. Furthermore, some of these proteins play a key role in immune modulation and are therefore therapeutic protein candidates.

Computational Biology↗

PubMed2004

VisANT: an online visualization and analysis tool for biological interaction data.

BACKGROUND: New techniques for determining relationships between biomolecules of all types--genes, proteins, noncoding DNA, metabolites and small molecules--are now making a substantial contribution to the widely discussed explosion of facts about the cell. The data generated by these techniques promote a picture of the cell as an interconnected information network, with molecular components linked with one another in topologies that can encode and represent many features of cellular function. This networked view of biology brings the potential for systematic understanding of living molecular systems. RESULTS: We present VisANT, an application for integrating biomolecular interaction data into a cohesive, graphical interface. This software features a multi-tiered architecture for data flexibility, separating back-end modules for data retrieval from a front-end visualization and analysis package. VisANT is a freely available, open-source tool for researchers, and offers an online interface for a large range of published data sets on biomolecular interactions, including those entered by users. This system is integrated with standard databases for organized annotation, including GenBank, KEGG and SwissProt. VisANT is a Java-based, platform-independent tool suitable for a wide range of biological applications, including studies of pathways, gene regulation and systems biology. CONCLUSION: VisANT has been developed to provide interactive visual mining of biological interaction data sets. The new software provides a general tool for mining and visualizing such data in the context of sequence, pathway, structure, and associated annotations. Interaction and predicted association data can be combined, overlaid, manipulated and analyzed using a variety of built-in functions. VisANT is available at http://visant.bu.edu.

Animals↗

PubMed2005

Ab initio identification of putative human transcription factor binding sites by comparative genomics.

BACKGROUND: Understanding transcriptional regulation of gene expression is one of the greatest challenges of modern molecular biology. A central role in this mechanism is played by transcription factors, which typically bind to specific, short DNA sequence motifs usually located in the upstream region of the regulated genes. We discuss here a simple and powerful approach for the ab initio identification of these cis-regulatory motifs. The method we present integrates several elements: human-mouse comparison, statistical analysis of genomic sequences and the concept of coregulation. We apply it to a complete scan of the human genome. RESULTS: By using the catalogue of conserved upstream sequences collected in the CORG database we construct sets of genes sharing the same overrepresented motif (short DNA sequence) in their upstream regions both in human and in mouse. We perform this construction for all possible motifs from 5 to 8 nucleotides in length and then filter the resulting sets looking for two types of evidence of coregulation: first, we analyze the Gene Ontology annotation of the genes in the set, searching for statistically significant common annotations; second, we analyze the expression profiles of the genes in the set as measured by microarray experiments, searching for evidence of coexpression. The sets which pass one or both filters are conjectured to contain a significant fraction of coregulated genes, and the upstream motifs characterizing the sets are thus good candidates to be the binding sites of the TF's involved in such regulation. In this way we find various known motifs and also some new candidate binding sites. CONCLUSION: We have discussed a new integrated algorithm for the "ab initio" identification of transcription factor binding sites in the human genome. The method is based on three ingredients: comparative genomics, overrepresentation, different types of coregulation. The method is applied to a full-scan of the human genome, giving satisfactory results.

Algorithms↗