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Results for “Sequence Alignment”

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PubMed2003

Cloning and expression of a new Tibetan hulless barley (Hordeum vulgare) beta-1,3-glucanase gene.

A new full-length beta-1,3-glucanase cDNA was obtained by RT-PCR and RACE techniques from Tibet hulless barley and its complete gene sequence obtained by DNA Walking. Sequence alignment with the BLAST program showed that cDNA has high similarity with barley beta-1,3-glucanase II. The gene was functionally expressed in E. coli and the recombinant protein catalysed the hydrolysis of beta-1,3-glucan with an action pattern characteristic of a beta-1,3-glucan endohydrolase (EC 3.2.1.39). Southern blot analysis indicated that the gene is a member of a small gene family. RT-PCR and northern blot analysis indicated it is constitutively expressed in barley shoots.

Amino Acid Sequence↗

PubMed2006

The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization.

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

Amino Acid Sequence↗

PubMed1996

Object-oriented sequence analysis: SCL--a C++ class library.

SCL (Sequence Class Library) is a class library written in the C++ programming language. Designed using object-oriented programming principles, SCL consists of classes of objects performing tasks typically needed for analyzing DNA or protein sequences. Among them are very flexible sequence classes, classes accessing databases in various formats, classes managing collections of sequences, as well as classes performing higher-level tasks like calculating a pairwise sequence alignment. SCL also includes classes that provide general programming support, like a dynamically growing array, sets, matrices, strings, classes performing file input/output, and utilities for error handling. By providing these components, SCL fosters an explorative programming style: experimenting with algorithms and alternative implementations is encouraged rather than punished. A description of SCL's overall structure as well as an overview of its classes is given. Important aspects of the work with SCL are discussed in the context of a sample program.

Algorithms↗

PubMed2004

Molecular cloning, nucleotide sequencing and characterization of the flagellin gene from isolates of urease-positive thermophilic Campylobacter.

A primer pair which was expected to generate an amplicon of the estimated size (approximately 1700 base pair (bp)) of the flaA gene for Campylobacter jejuni amplified products of approximately 1450 bp for 33 of the 44 isolates of urease-positive thermophilic Campylobacter (UPTC). The primer pair, however, failed to amplify fragments for 11 isolates of UPTC, for all of the 12 isolates of urease-negative C. lari and for one isolate of C. coli. Nevertheless, it successfully amplified fragments of approximately 1700 bp for five isolates of C. jejuni and for nine isolates of C. coli. Thus, the fragments of the flaA gene of UPTC were shorter than those of C. jejuni and C. coli. After PCR amplification and nucleotide sequencing of the flaA genes from five UPTC NCTC isolates, the putative open reading frames (ORFs) were found to range from 1461 to 1479 bp. The amino acid and nucleotide sequence alignments demonstrated that the PCR clones contained the flaA gene; however, our data indicated that this locus was markedly shorter in the UPTC organisms examined, as they were approximately 85 amino acid residues shorter, mainly corresponding to approximate residue numbers 390-470 of the large variable region of C. jejuni 81116. Heterogeneity was indicated in the molecular mass of the flagellin purified from the isolates examined. Flagellin of UPTC was demonstrated to be genotypically and phenotypically smaller than those of C. jejuni.

Amino Acid Sequence↗

PubMed2003

In silico study of breast cancer associated gene 3 using LION Target Engine and other tools.

Sequence analysis of individual targets is an important step in annotation and validation. As a test case, we investigated human breast cancer associated gene 3 (BCA3) with LION Target Engine and with other bioinformatics tools. LION Target Engine confirmed that the BCA3 gene is located on 11p15.4 and that the two most likely splice variants (lacking exon 3 and exons 3 and 5, respectively) exist. Based on our manual curation of sequence data, it is proposed that an additional variant (missing only exon 5) published in a public sequence repository, is a prediction artifact. A significant number of new orthologs were also identified, and these were the basis for a high-quality protein secondary structure prediction. Moreover, our research confirmed several distinct functional domains as described in earlier reports. Sequence conservation from multiple sequence alignments, splice variant identification, secondary structure predictions, and predicted phosphorylation sites suggest that the removal of interaction sites through alternative splicing might play a modulatory role in BCA3. This in silico approach shows the depth and relevance of an analysis that can be accomplished by including a variety of publicly available tools with an integrated and customizable life science informatics platform.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach.

To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.

Adenoviridae↗

PubMed2002

Scoring residue conservation.

The importance of a residue for maintaining the structure and function of a protein can usually be inferred from how conserved it appears in a multiple sequence alignment of that protein and its homologues. A reliable metric for quantifying residue conservation is desirable. Over the last two decades many such scores have been proposed, but none has emerged as a generally accepted standard. This work surveys the range of scores that biologists, biochemists, and, more recently, bioinformatics workers have developed, and reviews the intrinsic problems associated with developing and evaluating such a score. A general formula is proposed that may be used to compare the properties of different particular conservation scores or as a measure of conservation in its own right.

Amino Acids↗

PubMed2003

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania.

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.

Animals↗

PubMed1997

Scoring hidden Markov models.

MOTIVATION: Statistical sequence comparison techniques, such as hidden Markov models and generalized profiles, calculate the probability that a sequence was generated by a given model. Log-odds scoring is a means of evaluating this probability by comparing it to a null hypothesis, usually a simpler statistical model intended to represent the universe of sequences as a whole, rather than the group of interest. Such scoring leads to two immediate questions: what should the null model be, and what threshold of log-odds score should be deemed a match to the model. RESULTS: This paper analyses these two issues experimentally. Within the context of the Sequence Alignment and Modeling software suite (SAM), we consider a variety of null models and suitable thresholds. Additionally, we consider HMMer's log-odds scoring and SAM's original Z-scoring method. Among the null model choices, a simple looping null model that emits characters according to the geometric mean of the character probabilities in the columns modeled by the hidden Markov model (HMM) performs well or best across all four discrimination experiments.

Algorithms↗

PubMed2003

Isolation and expression of cold-regulated cDNA from Chinese cabbage (Brassica pekinensis).

It has been established that changes in gene expression occur during cold acclimation in a wide range of plants. Here we present a novel cDNA encoding a protein with a calculated molecular mass of 25 kDa, designated cor25, from Brassica pekinensis cv. Zaoshu 5 (Chinese cabbage) that was similar with a cold-regulated Arabidopsis thaliana L. gene, cor47. Protein sequence alignment showed that COR25 had 53.5% of identity with COR47 and 59.6% of identity with ERD10, an Arabidopsis thaliana L. early-responsive gene to dehydration stress. Northern blot analysis revealed that the cor25 expression was cold inducible. Moreover, the transcripts of the cor25 accumulated in plants in response to exogenous application of ABA and water stress. The possible functions of the cold-regulated gene and the mechanism for plants to cope with low temperature are discussed.

Amino Acid Sequence↗

PubMed2005

MollDE: a homology modeling framework you can click with.

UNLABELLED: Molecular Integrated Development Environment (MolIDE) is an integrated application designed to provide homology modeling tools and protocols under a uniform, user-friendly graphical interface. Its main purpose is to combine the most frequent modeling steps in a semi-automatic, interactive way, guiding the user from the target protein sequence to the final three-dimensional protein structure. The typical basic homology modeling process is composed of building sequence profiles of the target sequence family, secondary structure prediction, sequence alignment with PDB structures, assisted alignment editing, side-chain prediction and loop building. All of these steps are available through a graphical user interface. MolIDE's user-friendly and streamlined interactive modeling protocol allows the user to focus on the important modeling questions, hiding from the user the raw data generation and conversion steps. MolIDE was designed from the ground up as an open-source, cross-platform, extensible framework. This allows developers to integrate additional third-party programs to MolIDE. AVAILABILITY: http://dunbrack.fccc.edu/molide/molide.php CONTACT: rl_dunbrack@fccc.edu.

Algorithms↗

PubMed1997

Sequence and structure alignment of Paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin.

On the basis of the conservation of neuraminidase (N) active-site residues in influenza virus N and paramyxovirus hemagglutinin-neuraminidase (HN), it has been suggested that the three-dimensional (3D) structures of the globular heads of the two proteins are broadly similar. In this study, details of this structural similarity are worked out. Detailed multiple sequence alignment of paramyxovirus HN proteins and influenza virus N proteins was based on the schematic representation of the previously proposed structural similarity. This multiple sequence alignment of paramyxovirus HN proteins was used as an intermediate to align the morbillivirus hemagglutinin (H) proteins with neuraminidase. Hypothetical 3D structures were built for paramyxovirus HN and morbillivirus H, based on homology modelling. The locations of insertions and deletions, glycosylation sites, active-site residues, and disulfide bridges agree with the proposed 3D structure of HN and H of the Paramyxoviridae. Moreover, details of the modelled H protein predict previously undescribed enzymatic activity. This prediction was confirmed for rinderpest virus and peste des petits ruminants virus. The enzymatic activity was highly substrate specific, because sialic acid was released only from crude mucins isolated from bovine submaxillary glands. The enzymatic activity may indicate a general infection mechanism for respiratory viruses, and the active site may prove to be a new target for antiviral compounds.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of Lma-p54, a new epicuticular surface protein in the cockroach Leucophaea maderae (Dictyoptera, oxyhaloinae).

The epicuticular surface protein Lma-p54 is imbedded in the "cuticular waxes" which cover the abdominal surface of the adult Leucophaea maderae. Natural Lma-p54 was purified and the complete cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p54 was expressed in the adult abdominal epidermis and in the chemical sense organs of both sexes. Sequence alignment indicates that Lma-p54 is closely related to aspartic proteases (EC 3.4.23). However, there are critical amino acid substitutions at the level of the active site and, since no proteolytic activity was detected in the abdominal secretion, the protein is likely inactive. As an inactive aspartic protease, Lma-p54 is related to pregnancy-associated glycoproteins (PAGs) which still present a peptide-binding ability. According to recent experiments using whole tergal protein secretions, a role in intraspecific contact recognition was proposed for this surface protein.

Amino Acid Sequence↗

PubMed2003

Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.

Amino Acid Sequence↗

PubMed2004

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression.

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.

Amino Acid Sequence↗

PubMed2001

Crypt1, an active Ac-like transposon from the chestnut blight fungus, Cryphonectria parasitica.

A moderately repetitive element was identified previously in the nuclear genome of the chestnut blight fungus, Cryphonectria parasitica, and has been used as a probe for population studies of the fungus. We report here that the repetitive element is a class II transposon of the hAT family of Activator (Ac)-like transposable elements. The element, named Crypt1, has a size of 3563 bp, including 21-bp terminal inverted repeats. A unique 8-bp direct repeat sequence flanking Crypt1 was identified in each of three clones examined. A single large ORF with the potential to encode a putative transposase of 946 amino acid residues was deduced from the sequence of Crypt1. Based on amino acid sequence alignments, Crypt1 is most closely related to other Ac-like transposons of filamentous ascomycetes. A single transcript of approximately 3.0 kb was identified by Northern hybridization experiments from Crypt1-containing isolates, suggesting that Crypt1 is an active element. An isolate containing a single, possibly defective, copy of Crypt1 was identified in C. parasitica isolates from China; no Crypt1 transcript was identified in this isolate. Transposition of Crypt1 was inferred from Southern and inverse PCR analyses of C. parasitica isolates maintained in the laboratory, but transposition appears to be a rare event.

Amino Acid Sequence↗

PubMed2003

A new structural motif in the left terminal domain of large viroids identified by covariation analysis.

Sequence alignment of non-hammerhead viroids (Pospiviroidae) reveals that they can be divided into large and small viroids. Covariation analysis of these groups provides evidence for a rod-like secondary structure, but also for two proposed suboptimal structures, namely hairpin I and a bifurcation in the left terminus of large viroids. The strongest covariation within the branched T1 domain of all large Pospiviroidae indicates a new tertiary interaction, resulting in a double-pseudoknot within the (+) strand or an additional hairpin in the (-) strand. The structural element is expected to play a role in the viroid's life cycle.

Algorithms↗

PubMed2004

Profile-based detection of microRNA precursors in animal genomes.

MOTIVATION: MicroRNAs (miRNA) are essential 21-22 nt regulatory RNAs produced from larger hairpin-like precursors. Local sequence alignment tools such as BLAST are able to identify new members of known miRNA families, but not all of them. We set out to estimate how many new miRNAs could be recovered using a profile-based strategy such as that implemented in the ERPIN program. RESULTS: We constructed alignments for 18 miRNA families and performed ERPIN searches on animal genomes. Results were compared to those of a WU-BLAST search at the same E-value cutoff. The two combined approaches produced 265 new miRNA candidates that were not found in miRNA databases. About 17% of hits were ERPIN specific. They showed better structural characteristics than BLAST-specific hits and included interesting candidates such as members of the miR-17 cluster in Tetraodon. Profile-based RNA detection will be an important complement of similarity search programs in the completion of miRNA collections.

Algorithms↗