Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Molecular Sequence Annotation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 829 records · Page 46Linked to original sources
PubMed2006

Cloning and radiation hybrid mapping of bovine toll-like receptor-4 (TLR-4) signaling molecules.

Toll-like receptor (TLR)-4 is a transmembrane receptor for lipopolysaccharide, a highly pro-inflammatory component of the outer membrane of Gram-negative bacteria. To date, molecules of the TLR-4 signaling pathway have not been well characterized in cattle. The goal of this study was to clone and sequence the full-length coding regions of bovine genes involved in TLR-4 signaling including CASP8, IRAK1, LY96 (MD-2), TICAM2, TIRAP, TOLLIP and TRAF 6 and to position these genes, as well as MyD88 and TICAM1, on the bovine genome using radiation hybrid mapping. Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%. Overall, this work should assist in the assembly and annotation of the bovine genome sequence, the identification of variations in genes critically involved in host innate immunity, and facilitate the study of TLR-4 signaling pathways in cattle.

Adaptor Proteins, Signal Transducing↗
PubMed2005

In silico discovery of enzyme-substrate specificity-determining residue clusters.

The binding between an enzyme and its substrate is highly specific, despite the fact that many different enzymes show significant sequence and structure similarity. There must be, then, substrate specificity-determining residues that enable different enzymes to recognize their unique substrates. We reason that a coordinated, not independent, action of both conserved and non-conserved residues determine enzymatic activity and specificity. Here, we present a surface patch ranking (SPR) method for in silico discovery of substrate specificity-determining residue clusters by exploring both sequence conservation and correlated mutations. As case studies we apply SPR to several highly homologous enzymatic protein pairs, such as guanylyl versus adenylyl cyclases, lactate versus malate dehydrogenases, and trypsin versus chymotrypsin. Without using experimental data, we predict several single and multi-residue clusters that are consistent with previous mutagenesis experimental results. Most single-residue clusters are directly involved in enzyme-substrate interactions, whereas multi-residue clusters are vital for domain-domain and regulator-enzyme interactions, indicating their complementary role in specificity determination. These results demonstrate that SPR may help the selection of target residues for mutagenesis experiments and, thus, focus rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.

Adenylyl Cyclases↗
PubMed2006

Reverse transcriptase template switching and false alternative transcripts.

Reverse transcriptase (RT) can switch from one template to another in a homology-dependent manner. In the study of eukaryotic transcripts, this propensity of RT can produce an artificially deleted cDNA, which can be wrongly interpreted as an alternative transcript. Here, we have investigated the presence of such template-switching artifacts in cDNA databases, by scanning a collection of human splice sites (Information for the Coordinates of Exons, ICE database). We have confirmed several cases at the experimental level. Artifacts represent a significant portion of apparently spliced sequences using noncanonical splice signals but are rare in the context of the whole database. However, care should be taken in the annotation of alternative transcripts, especially when the RT used is poorly thermostable and when the putative intron is flanked by direct repeats, which are the substrate for template switching.

Alternative Splicing↗
PubMed2006

Using annotated peptide mass spectrum libraries for protein identification.

A system for creating a library of tandem mass spectra annotated with corresponding peptide sequences was described. This system was based on the annotated spectra currently available in the Global Proteome Machine Database (GPMDB). The library spectra were created by averaging together spectra that were annotated with the same peptide sequence, sequence modifications, and parent ion charge. The library was constructed so that experimental peptide tandem mass spectra could be compared with those in the library, resulting in a peptide sequence identification based on scoring the similarity of the experimental spectrum with the contents of the library. A software implementation that performs this type of library search was constructed and successfully used to obtain sequence identifications. The annotated tandem mass spectrum libraries for the Homo sapiens, Mus musculus, and Saccharomyces cerevisiae proteomes and search software were made available for download and use by other groups.

Amino Acid Sequence↗
PubMed2003

Stability of plasmid pA387 derivatives in Amycolatopsis mediterranei producing rifamycin.

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.

Actinomycetaceae↗
PubMed2003

Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin.

Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.

DNA Primers↗
PubMed2004

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence.

BACKGROUND: Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics. RESULTS: We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes. CONCLUSIONS: Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association.

Chromosome Mapping↗
PubMed2006

LocustDB: a relational database for the transcriptome and biology of the migratory locust (Locusta migratoria).

BACKGROUND: The migratory locust (Locusta migratoria) is an orthopteran pest and a representative member of hemimetabolous insects for biological studies. Its transcriptomic data provide invaluable information for molecular entomology and pave a way for the comparative research of other medically, agronomically, and ecologically relevant insects. We developed the first transcriptomic database of the locust (LocustDB), building necessary infrastructures to integrate, organize, and retrieve data that are either currently available or to be acquired in the future. DESCRIPTION: LocustDB currently hosts 45,474 high-quality EST sequences from the locust, which were assembled into 12,161 unigenes. It, through user-friendly web interfaces, allows investigators to freely access sequence data, including homologous/orthologous sequences, functional annotations, and pathway analysis, based on conserved orthologous groups (COG), gene ontology (GO), protein domain (InterPro), and functional pathways (KEGG). It also provides information from comparative analysis based on data from the migratory locust and five other invertebrate species, including the silkworm, the honeybee, the fruitfly, the mosquito and the nematode. The website address of LocustDB is http://locustdb.genomics.org.cn/. CONCLUSION: LocustDB starts with the first transcriptome information for an orthopteran and hemimetabolous insect and will be extended to provide a framework for incorporating in-coming genomic data of relevant insect groups and a workbench for cross-species comparative studies.

Animals↗
PubMed2005

Transcriptional slippage in bacteria: distribution in sequenced genomes and utilization in IS element gene expression.

BACKGROUND: Transcription slippage occurs on certain patterns of repeat mononucleotides, resulting in synthesis of a heterogeneous population of mRNAs. Individual mRNA molecules within this population differ in the number of nucleotides they contain that are not specified by the template. When transcriptional slippage occurs in a coding sequence, translation of the resulting mRNAs yields more than one protein product. Except where the products of the resulting mRNAs have distinct functions, transcription slippage occurring in a coding region is expected to be disadvantageous. This probably leads to selection against most slippage-prone sequences in coding regions. RESULTS: To find a length at which such selection is evident, we analyzed the distribution of repetitive runs of A and T of different lengths in 108 bacterial genomes. This length varies significantly among different bacteria, but in a large proportion of available genomes corresponds to nine nucleotides. Comparative sequence analysis of these genomes was used to identify occurrences of 9A and 9T transcriptional slippage-prone sequences used for gene expression. CONCLUSIONS: IS element genes are the largest group found to exploit this phenomenon. A number of genes with disrupted open reading frames (ORFs) have slippage-prone sequences at which transcriptional slippage would result in uninterrupted ORF restoration at the mRNA level. The ability of such genes to encode functional full-length protein products brings into question their annotation as pseudogenes and in these cases is pertinent to the significance of the term 'authentic frameshift' frequently assigned to such genes.

Adenosine↗
PubMed2003

How well is enzyme function conserved as a function of pairwise sequence identity?

Enzyme function conservation has been used to derive the threshold of sequence identity necessary to transfer function from a protein of known function to an unknown protein. Using pairwise sequence comparison, several studies suggested that when the sequence identity is above 40%, enzyme function is well conserved. In contrast, Rost argued that because of database bias, the results from such simple pairwise comparisons might be misleading. Thus, by grouping enzyme sequences into families based on sequence similarity and selecting representative sequences for comparison, he showed that enzyme function starts to diverge quickly when the sequence identity is below 70%. Here, we employ a strategy similar to Rost's to reduce the database bias; however, we classify enzyme families based not only on sequence similarity, but also on functional similarity, i.e. sequences in each family must have the same four digits or the same first three digits of the enzyme commission (EC) number. Furthermore, instead of selecting representative sequences for comparison, we calculate the function conservation of each enzyme family and then average the degree of enzyme function conservation across all enzyme families. Our analysis suggests that for functional transferability, 40% sequence identity can still be used as a confident threshold to transfer the first three digits of an EC number; however, to transfer all four digits of an EC number, above 60% sequence identity is needed to have at least 90% accuracy. Moreover, when PSI-BLAST is used, the magnitude of the E-value is found to be weakly correlated with the extent of enzyme function conservation in the third iteration of PSI-BLAST. As a result, functional annotation based on the E-values from PSI-BLAST should be used with caution. We also show that by employing an enzyme family-specific sequence identity threshold above which 100% functional conservation is required, functional inference of unknown sequences can be accurately accomplished. However, this comes at a cost: those true positive sequences below this threshold cannot be uniquely identified.

Animals↗
PubMed1997

The Signal Recognition Particle Database (SRPDB).

The SRPDB (Signal Recognition Particle Database) offers aligned SRP RNA and SRP protein sequences, phylogenetically ordered and annotated. This release adds three SRP RNA sequences (totaling 96 SRP RNA sequences) and 11 SRP protein sequences (a total of 39 protein sequences from SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 or SRP72). Also downloadable are sample SRP RNA secondary structure diagrams, a three-dimensional model of the human SRP RNA, search motifs and software.

Amino Acid Sequence↗
PubMed1998

The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis.

The completion of the entire 4.2-Mb genome sequence of the gram-positive bacterium Bacillus subtilis has been a milestone for biological studies on this model organism. This paper describes bioinformatics work related to this joint European and Japanese project: methods and strategies for gene annotation and detection of sequencing errors, using an integrated cooperative computer environment (Imagene); construction of a specialized database for data management and a WWW server for data retrieval (SubtiList); DNA sequence analysis, yielding striking results on oligonucleotide bias, repeated sequences, and codon usage, all landmarks of evolutionary events shaping the B. subtilis genome.

Amino Acid Sequence↗
PubMed2006

Phylogenetic supermatrix analysis of GenBank sequences from 2228 papilionoid legumes.

A comprehensive phylogeny of papilionoid legumes was inferred from sequences of 2228 taxa in GenBank release 147. A semiautomated analysis pipeline was constructed to download, parse, assemble, align, combine, and build trees from a pool of 11,881 sequences. Initial steps included all-against-all BLAST similarity searches coupled with assembly, using a novel strategy for building length-homogeneous primary sequence clusters. This was followed by a combination of global and local alignment protocols to build larger secondary clusters of locally aligned sequences, thus taking into account the dramatic differences in length of the heterogeneous coding and noncoding sequence data present in GenBank. Next, clusters were checked for the presence of duplicate genes and other potentially misleading sequences and examined for combinability with other clusters on the basis of taxon overlap. Finally, two supermatrices were constructed: a "sparse" matrix based on the primary clusters alone (1794 taxa x 53,977 characters), and a somewhat more "dense" matrix based on the secondary clusters (2228 taxa x 33,168 characters). Both matrices were very sparse, with 95% of their cells containing gaps or question marks. These were subjected to extensive heuristic parsimony analyses using deterministic and stochastic heuristics, including bootstrap analyses. A "reduced consensus" bootstrap analysis was also performed to detect cryptic signal in a subtree of the data set corresponding to a "backbone" phylogeny proposed in previous studies. Overall, the dense supermatrix appeared to provide much more satisfying results, indicated by better resolution of the bootstrap tree, excellent agreement with the backbone papilionoid tree in the reduced bootstrap consensus analysis, few problematic large polytomies in the strict consensus, and less fragmentation of conventionally recognized genera. Nevertheless, at lower taxonomic levels several problems were identified and diagnosed. A large number of methodological issues in supermatrix construction at this scale are discussed, including detection of annotation errors in GenBank sequences; the shortage of effective algorithms and software for local multiple sequence alignment; the difficulty of overcoming effects of fragmentation of data into nearly disjoint blocks in sparse supermatrices; and the lack of informative tools to assess confidence limits in very large trees.

Algorithms↗
PubMed2003

ESTs from the basidiomycete Schizophyllum commune grown on nitrogen-replete and nitrogen-limited media.

Lambda phage cDNA libraries were constructed using mRNAs from the basidiomycete Schizophyllum commune grown on media with high or low nitrogen concentrations. A total of 440 clones were sequenced, representing 373 distinct transcripts. Of these, 166 showed significant similarity to annotated genes in GenBank. Those that could be tentatively identified using BLAST searches were classified by function using the Gene Ontology (GO) database. Genes with products involved in cell-cycle processes were more frequent in the nitrogen-limited libraries, while genes with products involved in protein biosynthesis were more frequent in the nitrogen-replete library. Overall, clones showed much greater similarity to the one publicly available basidiomycete genome, Phanerochaete chrysosporium, than to any of the ascomycete genomes.

Bacteriophage lambda↗
PubMed2000

Rapid automatic detection and alignment of repeats in protein sequences.

Many large proteins have evolved by internal duplication and many internal sequence repeats correspond to functional and structural units. We have developed an automatic algorithm, RADAR, for segmenting a query sequence into repeats. The segmentation procedure has three steps: (i) repeat length is determined by the spacing between suboptimal self-alignment traces; (ii) repeat borders are optimized to yield a maximal integer number of repeats, and (iii) distant repeats are validated by iterative profile alignment. The method identifies short composition biased as well as gapped approximate repeats and complex repeat architectures involving many different types of repeats in the query sequence. No manual intervention and no prior assumptions on the number and length of repeats are required. Comparison to the Pfam-A database indicates good coverage, accurate alignments, and reasonable repeat borders. Screening the Swissprot database revealed 3,000 repeats not annotated in existing domain databases. A number of these repeats had been described in the literature but most were novel. This illustrates how in times when curated databases grapple with ever increasing backlogs, automatic (re)analysis of sequences provides an efficient way to capture this important information.

Algorithms↗
PubMed2004

The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions.

UNLABELLED: The epoxide hydrolases and haloalkane dehalogenases database (EH/HD) integrates sequence and structure of a highly diverse protein family, including mainly the Asp-hydrolases of EHs and HDs but also proteins, such as Ser-hydrolases non-heme peroxidases, prolyl iminopetidases and 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. A total of 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. AVAILABILITY: The EH/HD database is available at http://www.led.uni-stuttgart.de

Amino Acid Motifs↗
PubMed2005

Characterization of 954 bovine full-CDS cDNA sequences.

BACKGROUND: Genome assemblies rely on the existence of transcript sequence to stitch together contigs, verify assembly of whole genome shotgun reads, and annotate genes. Functional genomics studies also rely on transcript sequence to create expression microarrays or interpret digital tag data produced by methods such as Serial Analysis of Gene Expression (SAGE). Transcript sequence can be predicted based on reconstruction from overlapping expressed sequence tags (EST) that are obtained by single-pass sequencing of random cDNA clones, but these reconstructions are prone to errors caused by alternative splice forms, transcripts from gene families with related sequences, and expressed pseudogenes. These errors confound genome assembly and annotation. The most useful transcript sequences are derived by complete insert sequencing of clones containing the entire length, or at least the full protein coding sequence (CDS) portion, of the source mRNA. While the bovine genome sequencing initiative is nearing completion, there is currently a paucity of bovine full-CDS mRNA and protein sequence data to support bovine genome assembly and functional genomics studies. Consequently, the production of high-quality bovine full-CDS cDNA sequences will enhance the bovine genome assembly and functional studies of bovine genes and gene products. The goal of this investigation was to identify and characterize the full-CDS sequences of bovine transcripts from clones identified in non-full-length enriched cDNA libraries. In contrast to several recent full-length cDNA investigations, these full-CDS cDNAs were selected, sequenced, and annotated without the benefit of the target organism's genomic sequence, by using comparison of bovine EST sequence to existing human mRNA to identify likely full-CDS clones for full-length insert cDNA (FLIC) sequencing. RESULTS: The predicted bovine protein lengths, 5' UTR lengths, and Kozak consensus sequences from 954 bovine FLIC sequences (bFLICs; average length 1713 nt, representing 762 distinct loci) are all consistent with previously sequenced mammalian full-length transcripts. CONCLUSION: In most cases, the bFLICs span the entire CDS of the genes, providing the basis for creating predicted bovine protein sequences to support proteomics and comparative evolutionary research as well as functional genomics and genome annotation. The results demonstrate the utility of the comparative approach in obtaining predicted protein sequences in other species.

5' Untranslated Regions↗
PubMed2006

Expressed sequence tag analysis in tef (Eragrostis tef (Zucc) Trotter).

Tef (Eragrostis tef (Zucc.) Trotter) is the most important cereal crop in Ethiopia; however, there is very little DNA sequence information available for this species. Expressed sequence tags (ESTs) were generated from 4 cDNA libraries: seedling leaf, seedling root, and inflorescence of E. tef and seedling leaf of Eragrostis pilosa, a wild relative of E. tef. Clustering of 3603 sequences produced 530 clusters and 1890 singletons, resulting in 2420 tef unigenes. Approximately 3/4 of tef unigenes matched protein or nucleotide sequences in public databases. Annotation of unigenes associated 68% of the putative tef genes with gene ontology categories. Identification of the translated unigenes for conserved protein domains revealed 389 protein family domains (Pfam), the most frequent of which was protein kinase. A total of 170 ESTs containing simple sequence repeats (EST-SSRs) were identified and 80 EST-SSR markers were developed. In addition, 19 single-nucleotide polymorphism (SNP) and (or) insertion-deletion (indel) and 34 intron fragment length polymorphism (IFLP) markers were developed. The EST database and molecular markers generated in this study will be valuable resources for further tef genetic research.

Databases, Nucleic Acid↗