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 487 records · Page 27Linked to original sources
PubMed2003

The path from molecular indicators of exposure to describing dynamic biological systems in an aquatic organism: microarrays and the fathead minnow.

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the discovery of genes, indicative of environmental exposure, in the Agency's long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Novel cDNAs and coding sequences will be used in DNA microarray analyses for pattern identification of stressor-specific, differentially up- and down-regulated genes. The methods currently used to discover genes in this organism, for which few annotated nucleic acid sequences exist, are cDNA subtraction libraries, differential display, exploiting PCR primers for known genes of other members of the family Cyprinidae and use of degenerate PCR primers designed from regions of moderate protein homology. Single or multiple genes noted as being differentially expressed in microarray analyses will then be used in separate studies to measure bioavailable stressors in the laboratory and field. These analyses will be accomplished by quantitative RT-PCR. Moving from analysis of single gene exposures to the global state of the transcriptome offers possibilities that those genes identified by DNA microarray analyses might be critical components of dynamic biological systems and networks, wherein chemical stressors exert toxic effects through various modes of action. Additionally, the ability to discriminate bioavailability of stressors in complex environmental mixtures, and correlation with adverse effects downstream from these early molecular events, presents challenging new ground to be broken in the area of risk assessment.

Animals↗
PubMed2003

Reevaluating human gene annotation: a second-generation analysis of chromosome 22.

We report a second-generation gene annotation of human chromosome 22. Using expressed sequence databases, comparative sequence analysis, and experimental verification, we have extended genes, fused previously fragmented structures, and identified new genes. The total length in exons of annotation was increased by 74% over our previously published annotation and includes 546 protein-coding genes and 234 pseudogenes. Thirty-two potential protein-coding annotations are partial copies of other genes, and may represent duplications on an evolutionary path to change or loss of function. We also identified 31 non-protein-coding transcripts, including 16 possible antisense RNAs. By extrapolation, we estimate the human genome contains 29,000-36,000 protein-coding genes, 21,300 pseudogenes, and 1500 antisense RNAs. We suggest that our revised annotation criteria provide a paradigm for future annotation of the human genome.

Animals↗
PubMed2003

Network approaches to genome annotation and the challenge of Anopheles gambiae.

The rapid advancement of current sequencing projects has provided significant challenges for the swift and accurate processing, integration and analysis of biological data. Such challenges will only become greater as a new round of organisms, both well known and obscure, await sequencing and further study. While perhaps less well studied at the molecular level than Drosophila melanogaster, the recently sequenced mosquito, Anopheles gambiae, provides an example of what will be faced by many of these awaiting projects. This review presents a brief overview of some of the challenges associated with these future genome projects (including Anopheles) as well as a description of recent computational approaches that utilize genome context for the prediction of gene and/or protein function. Such approaches show promise not only for the study of Anopheles, but also in the study of biological processes in general.

Animals↗
PubMed2006

Facile recovery of individual high-molecular-weight, low-copy-number natural plasmids for genomic sequencing.

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

Chromosomes, Artificial, Bacterial↗
PubMed2005

Dasty and UniProt DAS: a perfect pair for protein feature visualization.

In this study, we present two freely available and complementary Distributed Annotation System (DAS) resources: a DAS reference server that provides up-to-date sequence and annotation from UniProt, with additional feature links and database cross-references from InterPro and a DAS client implemented using Java and Macromedia Flash that is optimized for the display of protein features.

Algorithms↗
PubMed2005

Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees.

In order to better understand the genetic regulation of secondary growth in hybrid aspen (Populus tremula L.xP. alba L.), we carried out a series of cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome analyses in vertical stem segments that represent a gradient of developmental stages with regard to secondary growth. This approach allowed us to screen >80% of the transcriptome expressed in six samples and identify genes differentially expressed with the progress of secondary growth, in a tissue-specific manner. Of the 76,800 transcript-derived fragments (TDFs) analyzed, 271 TDFs were selected and sequenced based on their differential expression patterns. Many of the xylem-up-regulated genes were involved in cell wall and lignin biosynthesis, while the bark-up-regulated genes had diverse functional roles. About 25% of the xylem-up-regulated TDFs analyzed were involved in the phenylpropanoid biosynthesis pathway, which produces the cell wall polymer lignin and various wood extractives. In addition, many of the TDFs showing secondary xylem-specific expression were annotated as genes not previously reported in Populus, including novel cell death proteins, cytoskeleton-interacting proteins, transporters and putative transcription factors.

Blotting, Northern↗
PubMed1999

Building dictionaries of 1D and 3D motifs by mining the Unaligned 1D sequences of 17 archaeal and bacterial genomes.

We have used the Teiresias algorithm to carry out unsupervised pattern discovery in a database containing the unaligned ORFs from the 17 publicly available complete archaeal and bacterial genomes and build a 1D dictionary of motifs. These motifs which we refer to as seqlets account for and cover 97.88% of this genomic input at the level of amino acid positions. Each of the seqlets in this 1D dictionary was located among the sequences in Release 38.0 of the Protein Data Bank and the structural fragments corresponding to each seqlet's instances were identified and aligned in three dimensions: those of the seqlets that resulted in RMSD errors below a pre-selected threshold of 2.5 Angstroms were entered in a 3D dictionary of structurally conserved seqlets. These two dictionaries can be thought of as cross-indices that facilitate the tackling of tasks such as automated functional annotation of genomic sequences, local homology identification, local structure characterization, comparative genomics, etc.

Algorithms↗
PubMed2003

Phylogenomic analysis of the Giardia intestinalis transcarboxylase reveals multiple instances of domain fusion and fission in the evolution of biotin-dependent enzymes.

Sequencing of the gene encoding a pyruvate carboxylase-like protein from the amitochondrial eukaryote Giardia intestinalis revealed a 1,338 aa protein composed of acetyl-CoA carboxyltransferase (ACCT), pyruvate carboxyltransferase (PycB), and biotin carboxyl carrier protein (BCCP) domains, linked in a single polypeptide chain. This particular domain combination has been previously seen only in the methylmalonyl-CoA:pyruvate transcarboxylase from Propionibacterium freudenreichii, where each of these domains is encoded by an individual gene and forms a separate subunit. To get an insight into the evolutionary origin and biochemical function of the G. intestinalis enzyme, we compared its domain composition to those of other biotin-dependent enzymes and performed a phylogenetic analysis of each of its domains. The results obtained indicate that: (1) evolution of the BCCP domain included several domain fusion events, leading to the ACCT-BCCP and PycB-BCCP domain combinations; (2) fusions of the PycB and BCCP domains in pyruvate carboxylases and oxaloacetate decarboxylases occurred on several independent occasions in different prokaryotic lineages, probably due to selective pressure towards co-expression of these genes, and (3) because newly sequenced biotin-dependent enzymes are often misannotated in sequence databases, their annotation as either carboxylases, decarboxylases, or transcarboxylases has to rely on detailed analysis of their domain composition, operon organization of the corresponding genes, gene content in the particular genome, and phylogenetic analysis.

Acetyl-CoA Carboxylase↗
PubMed2005

Genome cluster database. A sequence family analysis platform for Arabidopsis and rice.

The genome-wide protein sequences from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) spp. japonica were clustered into families using sequence similarity and domain-based clustering. The two fundamentally different methods resulted in separate cluster sets with complementary properties to compensate the limitations for accurate family analysis. Functional names for the identified families were assigned with an efficient computational approach that uses the description of the most common molecular function gene ontology node within each cluster. Subsequently, multiple alignments and phylogenetic trees were calculated for the assembled families. All clustering results and their underlying sequences were organized in the Web-accessible Genome Cluster Database (http://bioinfo.ucr.edu/projects/GCD) with rich interactive and user-friendly sequence family mining tools to facilitate the analysis of any given family of interest for the plant science community. An automated clustering pipeline ensures current information for future updates in the annotations of the two genomes and clustering improvements. The analysis allowed the first systematic identification of family and singlet proteins present in both organisms as well as those restricted to one of them. In addition, the established Web resources for mining these data provide a road map for future studies of the composition and structure of protein families between the two species.

Algorithms↗
PubMed2026

Application of a Translational Research Platform to Unveil Efficacy Signals and Mechanisms of Resistance of FGFR Inhibitors in Multiple FGFR-Altered Solid Tumors.

PURPOSE: The predictive value of fibroblast growth factor receptor (FGFR) amplifications (amp) and the role of FGFR mutations (mut) beyond known activating variants remain unclear. We aimed to establish a translational research platform to characterize FGFR alterations (alt) and explore their potential as predictive biomarkers for FGFR-targeted agents. EXPERIMENTAL DESIGN: This ambispective study included a retrospective analysis of patients with FGFR-alt tumors treated with selective FGFR inhibitors (FGFRi) and a prospective collection of longitudinal tumor samples. Patient-derived xenografts (PDX) were generated to investigate FGFRi mechanisms of action and resistance. Molecular characterization included genomic, transcriptomic, proteomic, and functional analyses using the Functional Annotation for Cancer Treatment (FACT) assay. RESULTS: Among 36 retrospectively analyzed patients, clinical benefit from FGFRis was observed in cases with FGFR mRNA overexpression or FGFR2/11q co-amp, but no association was found with the amplification levels. In archival tumor samples, exploratory proteomic analysis showed FGFR1-4 protein expression in 78% of FGFR1/2-amp tumors detected by fluorescence in situ hybridization. RNA sequencing identified a higher prevalence of FGFR mRNA overexpression than proteomic analysis. Among patients harboring FGFR-mut, only one bladder cancer with an FGFR3-mut S249C derived benefit. FACT assay supported the functional activity of selected variants, including FGFR3 T689M, and suggested potential resistance mechanisms involving PI3K/PTEN and MAPK pathway co-alterations. A prospective FGFR-alt PDX biorepository enabled exploratory biomarker analyses, supporting the hypothesis that FGFR1-4 mRNA expression may better reflect FGFR dependency than genomic alterations alone. CONCLUSIONS: These findings highlight the complexity of FGFR-driven oncogenesis and support integrative molecular approaches to refine patient selection for FGFR-targeted therapies.

Humans↗
PubMed2006

The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction.

The blood fluke Schistosoma mansoni can live for years in the hepatic portal system of its human host and so must possess very effective mechanisms of immune evasion. The key to understanding how these operate lies in defining the molecular organisation of the exposed parasite surface. The adult worm is covered by a syncytial tegument, bounded externally by a plasma membrane and overlain by a laminate secretion, the membranocalyx. In order to determine the protein composition of this surface, the membranes were detached using a freeze/thaw technique and enriched by sucrose density gradient centrifugation. The resulting preparation was sequentially extracted with three reagents of increasing solubilising power. The extracts were separated by 2-DE and their protein constituents were identified by MS/MS, yielding predominantly cytosolic, cytoskeletal and membrane-associated proteins, respectively. After extraction, the final pellet containing membrane-spanning proteins was processed by liquid chromatographic techniques before MS. Transporters for sugars, amino acids, ions and other solutes were found together with membrane enzymes and proteins concerned with membrane structure. The proteins identified were categorised by their function and putative location on the basis of their homology with annotated proteins in other organisms.

Animals↗
PubMed2001

Genomics, the cytoskeleton and motility.

The draft human genome sequence is an important step in cataloguing the molecular hardware that supports the processes of life. Here I look at what we have learned from the draft sequence about our cytoskeletal and motility systems. Most cytoskeletal and motility proteins were discovered previously by biochemical isolation, traditional cloning methods or random sequences of complementary DNAs. The ongoing challenges of assembling and annotating genes for motor proteins with long, fragmented coding sequences emphasize the importance of expert knowledge of related proteins and confirmatory evidence from cDNA sequences.

Actins↗
PubMed2003

Molecular characterization of prostatic small-cell neuroendocrine carcinoma.

OBJECTIVES: A subset of prostate carcinomas is composed predominantly, even exclusively, of neuroendocrine (NE) cells. In this report, we sought to characterize the gene expression profile of a prostate small cell NE carcinoma by assessing the diversity and abundance of transcripts in the LuCaP 49 prostate small cell carcinoma xenograft. METHODS: We constructed a cDNA library (PRCA3) from the LuCap 49 prostate small cell xenograft. Single pass DNA sequencing of randomly selected cDNA clones followed by sequence assembly and annotation produced a library of Expressed Sequence Tags (ESTs) representing the LuCaP 49 transcriptome. Comparative sequence analysis with ESTs derived from prostate adenocarcinoma libraries was performed using statistical algorithms designed to identify differentially expressed sequences. Putative NE cell-specific genes were further examined by Northern analysis. RESULTS: Sequence assembly and analysis identified 1,447 distinct genes expressed in the LuCaP 49 cDNA library. These include cDNAs encoding the NE markers secretogranin (SCG2), CD24, and ENO2. Northern analysis revealed that three additional genes, ASCL1, INA, and SV2B are expressed in LuCaP 49 but not in various prostate cancer cell lines or xenografts. Fifteen genes were identified with a statistical probability (P > 0.9) of being up-regulated in LuCaP 49 small cell carcinoma relative to prostate adenocarcinoma (two primary prostate adenocarcinomas and the LNCaP prostate adenocarcinoma cell line). CONCLUSIONS: Prostate small cell carcinoma expresses a diverse repertoire of genes that reflect characteristics of their NE cell of origin. ASCL1, INA, and SV2B are potential molecular markers for small cell NE tumors and NE cells of the prostate. This small cell NE carcinoma gene expression profile may yield insights into the development, progression, and treatment of subtypes of prostate cancer.

Aged↗
PubMed2003

Molecular cloning and RNA expression of a novel Drosophila calpain, Calpain C.

The calpains are Ca(2+)-activated cysteine proteases whose biochemical properties have been extensively characterized in vitro. Less is known, however, about the physiological role of calpains. In this respect, Drosophila melanogaster is a useful experimental organism to study calpain activity and regulation in vivo. The sequencing of the fly genome has been recently completed and a novel calpain homologue has been identified in the CG3692 gene product. We embarked on the cloning and characterization of this putative novel calpain. We demonstrate that the actual calpain is different from the predicted protein and we provide experimental evidence for the correction of the genomic annotation. This novel protein, Calpain C, must be catalytically inactive, having mutated active site residues but is otherwise structurally similar to the other known fly calpains. Moreover, we analysed Calpain C RNA expression during Drosophila development by RT-PCR and RNA in situ hybridization, which revealed strong expression in the salivary glands.

Amino Acid Sequence↗
PubMed2004

Characterization and comparative analyses of transcriptomes from the normal and neoplastic human prostate.

BACKGROUND: The prostate gland is a highly specialized organ with functional attributes that serve to enhance the fertility of mammalian species. Pathological processes affecting the prostate include benign prostate hypertrophy and prostate carcinoma; diseases that account for major morbidity and mortality in middle-aged and elderly men. To facilitate studies of biological processes uniquely represented in the prostate and assess molecular alterations associated with prostate carcinoma, we sought to establish the diversity of gene expression in the normal and neoplastic prostate through the compilation and analysis of a prostate transcriptome. METHODS: We assembled and annotated ESTs derived from prostate cDNA libraries that were either produced in our laboratory or available from public sequence repositories such as CGAP, dbEST, and Unigene. Determinations of differential gene expression between the normal prostate, other normal tissues, and neoplastic prostate tissues was performed using statistical algorithms. Confirmation of differential expression was performed by quantitative PCR and Northern analysis. RESULTS: A total of 99,448 high-quality ESTs were assembled and annotated to produce a prostate transcriptome comprised of 24,580 distinct TUs. Comparative analyses of gene expression levels identified 61 TUs with exclusive expression in the prostate and 45 TUs with high levels of expression in the prostate relative to at least 25 other normal tissues (P > 0.99). Comparative analyses of ESTs derived from neoplastic prostate tissues identified 75 genes with dysregulated expression in cancer (P > 0.99). CONCLUSIONS: The human prostate expresses a diverse repertoire of genes that reflect a functionally complex organ. The identification of genes with prostate-restricted or enhanced expression may provide additional insights into the biochemical processes that interact to form the developmental, signaling, and metabolic pathways of the normal and neoplastic gland.

Algorithms↗
PubMed2004

Identification and phenotypic characterization of the cell-division protein CdpA.

Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5'-region. However, a strong putative terminator with a free energy of -16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell-cell separation and designated the gene as cell-division protein, cdpA.

Bacterial Adhesion↗
PubMed2000

The physics of DNA and the annotation of the Plasmodium falciparum genome.

A gene identification procedure is formulated, based on large-scale structural analyses of genomic sequences. The structural property is the physical - thermal - stability of the DNA double-helix, as described by the classical helix-coil model. The analyses are detailed for the Plasmodium falciparum genome, which represents one of the most difficult cases for the gene identification problem (notably because of the extreme AT-richness of the genome). In this genome, the coding domains (either uninterrupted genes or exons in split genes) are accurately identified as regions of high thermal stability. The conclusion is based on the study of the available cloned genes, of which 17 examples are described in detail. These examples demonstrate that the physical criterion is valid for the detection of coding regions whose lengths extend from a few base pairs up to several thousand base pairs. Accordingly, the structural analyses can provide a powerful and convenient tool for the identification of complex genes in the P. falciparum genome. The limits of such a scheme are discussed. The gene identification procedure is applied to the completely sequenced chromosomes (2 and 3), and the results are compared with the database annotations. The structural analyses suggest more or less extensive revision to the annotations, and also allow new putative genes to be identified in the chromosome sequences. Several examples of such new genes are described in detail.

Animals↗
PubMed1997

Zooming in on the human-mouse comparative map: genome conservation re-examined on a high-resolution scale.

Over the past decade, conservation of genetic linkage groups has been shown in mammals and used to great advantage, fueling significant exchanges of gene mapping and functional information especially between the genomes of humans and mice. As human physical maps increase in resolution from chromosome bands to nucleotide sequence, comparative alignments of mouse and human regions have revealed striking similarities and surprising differences between the genomes of these two best-mapped mammalian species. Whereas, at present, very few mouse and human regions have been compared on the physical level, existing studies provide intriguing insights to genome evolution, including the observation of recent duplications and deletions of genes that may play significant roles in defining some of the biological differences between the two species. Although high-resolution conserved marker-based maps are currently available only for human and mouse, a variety of new methods and resources are speeding the development of comparative maps of additional organisms. These advances mark the first step toward establishment of the human genome as a reference map for vertebrate species, providing evolutionary and functional annotation to human sequence and vast new resources for genetic analysis of a variety of commercially, medically, and ecologically important animal models.

Animals↗