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Results for “Molecular Sequence Annotation”

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PubMed2000

Assignment of enzyme substrate specificity by principal component analysis of aligned protein sequences: an experimental test using DNA glycosylase homologs.

We have studied the relationship between amino acid sequence and substrate specificity in a DNA glycosylase family by characterizing experimentally the specificity of four new members of the family. We show that principal component analysis (PCA) of the sequence family correctly predicts the substrate specificity of one of the novel homologs even though conventional sequence analysis methods fail to group this homolog with other sequences of the same specificity. PCA also suggested, correctly, that another homolog characterized previously differs in its specificity from those sequences with which it clusters by conventional criteria. These results suggest that principal component analysis of sequence families can be a useful tool in annotating genome sequences when there is ambiguity concerning which subfamily a new homolog belongs to. Published 2000 Wiley-Liss, Inc.

Amino Acid Sequence↗
PubMed2005

Use of search algorithms to define specificity in Rab GTPase domain function.

The continuing explosion of sequencing data has inspired a corresponding effort in the annotation and classification of protein families. Within a particular protein family, however, individual members may have distinct functions, although they share a common fold and broadly defined physiological role. Rab GTPases are the largest subfamily of the Ras superfamily, yet from early in their discovery, it was apparent that each Rab protein has a unique subcellular localization and regulates a particular stage(s) membrane traffic. To gain insight into the contribution of individual residues to unique protein functions a general strategy is outlined. This method should allow the cell and molecular biologist with no specialist expertise to implement an algorithm that makes use of a combination of experimental and phylogenetic data. The algorithm is applicable to the analysis of any protein domain and here is illustrated with the analysis of residues contributing to the individual functions of a pair of Rab GTPases.

Algorithms↗
PubMed2025

Assembling genomes of non-model plants: A case study with evolutionary insights from Ranunculus (Ranunculaceae).

Whereas genome sequencing and assembly technologies are improving, cost can still be prohibitive for plant species with large, complex genomes. As a consequence, genomics work on some taxa in evolutionarily pivotal positions in the vascular plant tree of life has been hampered. The species-rich genus Ranunculus (Ranunculaceae) is an important angiosperm group for the study of polyploidy, apomixis, and reticulate evolution. However, neither mitochondrial nor high-quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short-read, Oxford Nanopore Technology (ONT) and PacBio (HiFi) long-read, and hybrid-read assembly strategies. We sequenced the diploid progenitor species R. cassubicifolius (R. auricomus species complex) and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156 kbp, 85 genes) and mitogenome (1.18 Mbp, 40 genes) sequences using Illumina and Illumina-PacBio-hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA, ycf15, or rps) with evolutionary implications. For the nuclear genome sequence, we favored a PacBio-based assembly polished three times with filtered short reads and subsequently scaffolded into eight pseudochromosomes by chromatin conformation data (Hi-C). We obtained a haploid genome sequence of 2.69 Gbp, with 94.1% complete BUSCO genes found and 35 482 annotated genes, and inferred ancient gene duplications compared to existing Ranunculales genomes. The genomic information presented here will enable advanced evolutionary-functional analyses for the species complex, but also for the genus and beyond Ranunculaceae.

Ranunculus↗
PubMed2025

Transposable elements are dysregulated in brains of individuals with major depressive disorder.

Transposable elements (TEs) are repetitive DNA sequences capable of being transcribed and re-integrated, or transposed, into distinct loci throughout the genome. While thought to be largely transcriptionally silenced in brain, TE transcription is increasingly recognized as dynamic and involved in human health and disease states, including in disorders of the brain. In this study, we annotated TE transcripts in publicly available RNA sequencing (RNAseq) of postmortem human brain tissue to investigate the expression profile of TE transcripts in individuals with major depressive disorder (MDD) compared to healthy controls. Our findings reveal a robust impact to TE transcript expression in the brains of subjects with MDD relative to controls. This work points to the aberrant transcription of cortical TEs as a potentially overlooked molecular signature of MDD.

Humans↗
PubMed1997

The uRNA database.

The uRNADB offers aligned, annotated and phylogenetically ordered sequences of several U RNAs. New to this release are RNAs from U7 (14 sequences), U8 (two sequences), U11 (three sequences), U12 (two sequences), U14 (11 sequences), U18, U48 and U49. A total of 34 new sequences were aligned with the previously compiled snRNAs U1, U2, U3, U4, U5 and U6.

Animals↗
PubMed2003

FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa.

Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

Amino Acid Sequence↗
PubMed2003

ExPASy: The proteomics server for in-depth protein knowledge and analysis.

The ExPASy (the Expert Protein Analysis System) World Wide Web server (http://www.expasy.org), is provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics (SIB). It provides access to a variety of databases and analytical tools dedicated to proteins and proteomics. ExPASy databases include SWISS-PROT and TrEMBL, SWISS-2DPAGE, PROSITE, ENZYME and the SWISS-MODEL repository. Analysis tools are available for specific tasks relevant to proteomics, similarity searches, pattern and profile searches, post-translational modification prediction, topology prediction, primary, secondary and tertiary structure analysis and sequence alignment. These databases and tools are tightly interlinked: a special emphasis is placed on integration of database entries with related resources developed at the SIB and elsewhere, and the proteomics tools have been designed to read the annotations in SWISS-PROT in order to enhance their predictions. ExPASy started to operate in 1993, as the first WWW server in the field of life sciences. In addition to the main site in Switzerland, seven mirror sites in different continents currently serve the user community.

Databases, Protein↗
PubMed2006

The proteins of human chromosome 21.

Recent genomic sequence annotation suggests that the long arm of human chromosome 21 encodes more than 400 genes. Because there is no evidence to exclude any significant segment of 21 q from containing genes relevant to the Down syndrome (DS) cognitive phenotype, all genes in this entire set must be considered as candidates. Only a subset, however, is likely to make critical contributions. Determining which these are is both a major focus in biology and a critical step in efficient development of therapeutics. The subtle molecular abnormality in DS, the 50% increase in chromosome 21 gene expression, presents significant challenges for researchers in detection and quantitation. Another challenge is the current limitation in understanding gene functions and in interpreting biological characteristics. Here, we review information on chromosome 21-encoded proteins compiled from the literature and from genomics and proteomics databases. For each protein, we summarize their evolutionary conservation, the complexity of their known protein interactions and their level of expression in brain, and discuss the implications and limitations of these data. For a subset, we discuss neurologically relevant phenotypes of mouse models that include knockouts, mutations, or overexpression. Lastly, we highlight a small number of genes for which recent evidence suggests a function in biochemical/cellular pathways that are relevant to cognition. Until knowledge deficits are overcome, we suggest that effective development of gene-phenotype correlations in DS requires a serious and continuous effort to assimilate broad categories of information on chromosome 21 genes, plus the creation of more versatile mouse models.

Animals↗
PubMed2002

FANTOM DB: database of Functional Annotation of RIKEN Mouse cDNA Clones.

FANTOM DB, the database of Functional Annotation of RIKEN Mouse cDNA Clones, is designed to store sequence information of RIKEN full-length enriched mouse cDNA clones, graphical views of sequence analysis results, curated functional annotation information and additional descriptions, including Gene Ontology terms. RIKEN's Mouse Gene Encyclopedia Project aims to collect full-length enriched cDNA clones from various mouse tissues, determine the full-length nucleotide sequences, infer their chromosomal locations by computer and characterize gene expression patterns. FANTOM DB has been developed to facilitate this work and to facilitate functional genomic studies such as positional candidate cloning, cDNA microarrays and protein interaction analyses. FANTOM DB contains 21 076 full-length cDNA sequences with rich functional annotations and is publicly available. FANTOM DB thus provides curated functional annotation to RIKEN full-length enriched mouse clones, and has links to other public resources. FANTOM DB can be accessed at http://fantom.gsc.riken.go.jp/db/.

Animals↗
PubMed2006

Robust analysis of 5'-transcript ends (5'-RATE): a novel technique for transcriptome analysis and genome annotation.

Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5'-transcript ends (5'-RATE) for rapid and cost-effective isolation of long 5' transcript ends (approximately 80 bp). It consists of three major steps including 5'-oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5'-RATE library revealed complex alternative transcription start sites and a 5' poly(A) tail in maize transcripts. Our results demonstrate that 5'-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

5' Untranslated Regions↗
PubMed2004

Whole genome sequence comparisons and "full-length" cDNA sequences: a combined approach to evaluate and improve Arabidopsis genome annotation.

To evaluate the existing annotation of the Arabidopsis genome further, we generated a collection of evolutionary conserved regions (ecores) between Arabidopsis and rice. The ecore analysis provides evidence that the gene catalog of Arabidopsis is not yet complete, and that a number of these annotations require re-examination. To improve the Arabidopsis genome annotation further, we used a novel "full-length" enriched cDNA collection prepared from several tissues. An additional 1931 genes were covered by new "full-length" cDNA sequences, raising the number of annotated genes with a corresponding "full-length" cDNA sequence to about 14,000. Detailed comparisons between these "full-length" cDNA sequences and annotated genes show that this resource is very helpful in determining the correct structure of genes, in particular, those not yet supported by "full-length" cDNAs. In addition, a total of 326 genomic regions not included previously in the Arabidopsis genome annotation were detected by this cDNA resource, providing clues for new gene discovery. Because, as expected, the two data sets only partially overlap, their combination produces very useful information for improving the Arabidopsis genome annotation.

Arabidopsis↗
PubMed2005

Distribution and intensity of constraint in mammalian genomic sequence.

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures approximately 3.9 neutral substitutions per site and spans approximately 1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering approximately 5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as "rejected substitutions". Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.

Animals↗
PubMed2005

Filtering high-throughput protein-protein interaction data using a combination of genomic features.

BACKGROUND: Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. RESULTS: In this study, we use a combination of 3 genomic features -- structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology -- as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90%) and good specificity (63%). We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. CONCLUSION: A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

Animals↗
PubMed2005

Complete nucleotide sequence of pSCV50, the virulence plasmid of Salmonella enterica serovar Choleraesuis SC-B67.

We carried out comparative analysis on the sequences of two 50-kb virulence plasmids of Salmonella enterica serovar Choleraesuis strains SC-B67 (pSCV50) and RF-1 (pKDSC50). The two plasmids share over 99% sequence similarity. Ninety-two nucleotide variations at 42 sites were detected between the two plasmids; pSCV50 contains 24 nucleotide substitutions, 6 deletions, and 62 insertions, compared to pKDSC50. Two regions in pSCV50 appeared to be more susceptible to changes: one is the non-virulence-associated transfer region (27.5-33.0 K) and the other a function-unknown region (9.0-10.5 K). We re-annotated pSCV50 using more advanced tools and the up-to-date databases and corrected the inaccurate annotation in pKDSC50. The results indicate that virulence-related genes on the 50-kb plasmid are under negative selection, suggesting that they play important roles in the expression of virulence during the process of infection, while other genes in this plasmid tend to evolve neutrally.

Chromosome Mapping↗
PubMed2004

Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to approximately 1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

Alu Elements↗
PubMed2002

MODBASE, a database of annotated comparative protein structure models.

MODBASE (http://guitar.rockefeller.edu/modbase) is a relational database of annotated comparative protein structure models for all available protein sequences matched to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies on PSI-BLAST, IMPALA and MODELLER. MODBASE uses the MySQL relational database management system for flexible and efficient querying, and the MODVIEW Netscape plugin for viewing and manipulating multiple sequences and structures. It is updated regularly to reflect the growth of the protein sequence and structure databases, as well as improvements in the software for calculating the models. For ease of access, MODBASE is organized into different datasets. The largest dataset contains models for domains in 304 517 out of 539 171 unique protein sequences in the complete TrEMBL database (23 March 2001); only models based on significant alignments (PSI-BLAST E-value < 10(-4)) and models assessed to have the correct fold are included. Other datasets include models for target selection and structure-based annotation by the New York Structural Genomics Research Consortium, models for prediction of genes in the Drosophila melanogaster genome, models for structure determination of several ribosomal particles and models calculated by the MODWEB comparative modeling web server.

Animals↗
PubMed2002

Differential gene expression analysis during porcine hepatocyte spheroid formation.

Primary porcine hepatocytes cultured in suspension self-assemble into multicellular aggregates or spheroids that display enhanced liver-specific functional capability and remain viable for an extended period of time in vitro. The molecular events underlying the process of spheroid formation were explored by differential gene expression analysis. Critical time points in spheroid formation were first identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of stress-related gene expression levels at different stages of spheroid formation. Suppression subtractive hybridization was used to identify transcripts up- or down-regulated at different stages of spheroid formation. Subsequently, three sets of reciprocal subtractions, comparing freshly isolated hepatocytes, spheroid-forming hepatocytes, and mature spheroids were carried out, and differentially expressed transcripts were isolated, cloned, sequenced, and annotated. A total of 65 genes and 14 novel transcripts were identified as differentially expressed, and very high sequence conservation between pig and human transcripts was observed. The resultant expressed sequence tags (ESTs) revealed a rapid decrease in the transcript levels of a subset of liver-specific genes, cytochrome P450s, and enzymes involved in heme biosynthesis, as well as up-regulation of genes involved in calcium-dependent vesicle trafficking and a number of acute-phase proteins in mature spheroids. Previous morphological and functional data on hepatocyte spheroid formation support cellular polarization of the hepatocyte into apical and basolateral domains in spheroids. This is important for the re-emergence of differentiated functions in vitro and is reflected by differences in gene expression patterns.

Animals↗
PubMed1995

PdbAlign, PdbDist and DistAlign: tools to aid in relating sequence variability to structure.

Many sequence analysis problems involve consideration of a multiple sequence alignment where the 3-dimensional structure of one (or more) of the aligned sequences is known. In such cases, it is useful to map the sequence variability onto the atomic co-ordinates of known structure. If the structure also includes a bound ligand (or the location of the active site is known), each column position in the multiple sequence alignment may be annotated with its 'distance' from the binding site. These annotations, together with a measure of sequence variability, provide additional insights into drug specificity, for example among viral mutants. This paper describes several useful programs that automate this analysis.

Amino Acid Sequence↗