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Results for “Sequence Alignment”

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PubMed2005

Four-helix bundle: a ubiquitous sensory module in prokaryotic signal transduction.

MOTIVATION: Transmembrane chemoreceptors in Escherichia coli utilize ligand-binding domains for detecting various external signals. The structure of this domain in the E.coli aspartate receptor, Tar, is known and its signal transduction mechanism is under investigation. Current domain models for this important sensory module are inaccurate and, therefore, cannot reveal the distribution of this domain within the current genomic landscape. RESULTS: We carried out sensitive and exhaustive PSI-BLAST searches initiated with the sequence corresponding to a known structure of the four-helix, ligand-binding domain of the aspartate chemoreceptor. From the resulting sequences, we built a multiple sequence alignment for this domain family, which confirmed that the current TarH model is erroneous and fails to detect most of the domain homologs. In the process, we developed a technique that visualizes the secondary structure prediction of each protein sequence in order to improve the multiple sequence alignment. We found that the four-helix up-and-down bundle represents a large domain family and includes representatives of all major classes of prokaryotic signal transduction, namely histidine kinases, di-guanylate cyclases and chemotaxis receptors.

Amino Acid Sequence↗

PubMed1994

The 16S/23S ribosomal spacer region of Coxiella burnetti.

The 16S/23S spacer region of Coxiella burnetti isolate Nine Nile, phase 1, was sequenced. Sequence analysis revealed two tRNA coding regions for tRNA(Ile) and tRNA(Ala). DNA sequence alignment demonstrated significant homology with tRNA species from Pseudomonas aeruginosa and Rhodobacter sphaeroides, respectively. The non-coding tRNA spacer region was unique to Coxiella burnetti, based on database alignment.

Cloning, Molecular↗

PubMed1997

Comparative sequence analysis of ribonucleases HII, III, II PH and D.

Escherichia coli ribonucleases (RNases) HII, III, II, PH and D have been used to characterise new and known viral, bacterial, archaeal and eucaryotic sequences similar to these endo- (HII and III) and exoribonucleases (II, PH and D). Statistical models, hidden Markov models (HMMs), were created for the RNase HII, III, II and PH and D families as well as a double-stranded RNA binding domain present in RNase III. Results suggest that the RNase D family, which includes Werner syndrome protein and the 100 kDa antigenic component of the human polymyositis scleroderma (PMSCL) autoantigen, is a 3'-->5' exoribonuclease structurally and functionally related to the 3'-->5' exodeoxyribonuclease domain of DNA polymerases. Polynucleotide phosphorylases and the RNase PH family, which includes the 75 kDa PMSCL autoantigen, possess a common domain suggesting similar structures and mechanisms of action for these 3'-->5' phosphorolytic enzymes. Examination of HMM-generated multiple sequences alignments for each family suggest amino acids that may be important for their structure, substrate binding and/or catalysis.

Amino Acid Sequence↗

PubMed2005

AMPDB: the Arabidopsis Mitochondrial Protein Database.

The Arabidopsis Mitochondrial Protein Database is an Internet-accessible relational database containing information on the predicted and experimentally confirmed protein complement of mitochondria from the model plant Arabidopsis thaliana (http://www.ampdb.bcs.uwa.edu.au/). The database was formed using the total non-redundant nuclear and organelle encoded sets of protein sequences and allows relational searching of published proteomic analyses of Arabidopsis mitochondrial samples, a set of predictions from six independent subcellular-targeting prediction programs, and orthology predictions based on pairwise comparison of the Arabidopsis protein set with known yeast and human mitochondrial proteins and with the proteome of Rickettsia. A variety of precomputed physical-biochemical parameters are also searchable as well as a more detailed breakdown of mass spectral data produced from our proteomic analysis of Arabidopsis mitochondria. It contains hyperlinks to other Arabidopsis genomic resources (MIPS, TIGR and TAIR), which provide rapid access to changing gene models as well as hyperlinks to T-DNA insertion resources, Massively Parallel Signature Sequencing (MPSS) and Genome Tiling Array data and a variety of other Arabidopsis online resources. It also incorporates basic analysis tools built into the query structure such as a BLAST facility and tools for protein sequence alignments for convenient analysis of queried results.

Arabidopsis Proteins↗

PubMed1998

Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme.

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

Amino Acid Sequence↗

PubMed2004

Isolation of mak1 from Actinoplanes missouriensis and evidence that Pep2 from Streptomyces coelicolor is a maltokinase.

The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor. Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria. More than 50% of the aligned sequences are longer than the mak1 gene from A. missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases. A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases. Transformation of S. lividans with plasmid vectors containing either the mak1 gene from A. missouriensis or the pep2 gene from S. coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity. The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized. The possible physiological function of maltokinases is discussed.

Amino Acid Sequence↗

PubMed2004

Phylogenetic analyses of the Fusarium poae, Fusarium sporotrichioides and Fusarium langsethiae species complex based on partial sequences of the translation elongation factor-1 alpha gene.

Phylogenetic relationships between four Fusarium species were studied using parts of the nuclear translation elongation factor-1 alpha (EF-1alpha) gene as a phylogenetic marker. Sequences from 12 isolates of Fusarium poae, 10 isolates of Fusarium sporotrichioides and 12 isolates of Fusarium langsethiae yielded 4, 5 and 5 haplotypes, respectively. In addition, we included one isolate of Fusarium kyushuense. The aligned sequences were subjected to neighbor-joining (NJ), maximum parsimony and maximum likelihood (ML) analyses. The results from the different analyses were highly concordant. The EF-1alpha-based phylogenies support the classification of F. langsethiae as a separate taxon in the section Sporotrichiella of Fusarium, as the closest sister taxon to F. sporotrichioides, while F. kyushuense is the sister taxon to F. poae. This corresponds well with the ability of F. langsethiae and F. sporotrichioides to produce T-2 and HT-2 toxins. In contrast, morphological characters indicate a closer relationship between F. langsethiae and F. poae on the one hand, and between F. sporotrichioides and F. kyushuense on the other hand.

DNA, Fungal↗

PubMed2001

Properties of the C-terminal domain of 4.1 proteins.

At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.

Amino Acid Sequence↗

PubMed1996

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy.

The growth factor receptor-bound protein-2 (Grb-2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly 13C-/15N-enriched protein as well as the protein containing selectively 15N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, 1H alpha, 13C alpha, 13C beta and 13CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423-438]. The core structure of the SH2 domain contains an antiparallel beta-sheet and two alpha-helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Structural similarities between the N-terminal domain of Clostridium pasteurianum hydrogenase and plant-type ferredoxins.

An N-terminal domain of Clostridium pasteurianum hydrogenase I, encompassing 76 residues out of the 574 composing the full-size enzyme, had previously been overproduced in Escherichia coli and shown to form a stable fold around a [2Fe-2S] cluster. This domain displays only marginal sequence similarity with [2Fe-2S] proteins of known structure, and therefore, two-dimensional 1H NMR has been implemented to elucidate features of the polypeptide fold. Despite the perturbing presence of the paramagnetic [2Fe-2S] cluster, 57 spin systems were detected in the TOCSY spectra, 52 of which were sequentially assigned through NOE connectivities. Several secondary structure elements were identified. The N terminus of the protein consists of two antiparallel beta strands followed by an alpha helix contacting both strands. Two additional antiparallel beta strands, one of them at the C terminus of the sequence, form a four-stranded beta sheet together with the two N-terminal strands. The proton resonances that can be attributed to this beta2alphabeta2 structural motif undergo no paramagnetic perturbations, suggesting that it is distant from the [2Fe-2S] cluster. In plant- and mammalian-type ferredoxins, a very similar structural pattern is found in the part of the protein farthest from the [2Fe-2S] cluster. This indicates that the N-terminal domain of C. pasteurianum hydrogenase folds in a manner very similar to those of plant- and mammalian-type ferredoxins over a significant part (ca. 50%) of its structure. Even in the vicinity of the metal site, where 1H NMR data are blurred by paramagnetic interactions, the N-terminal domains of hydrogenase and mammalian- and plant-type ferredoxins most likely display significant structural similarity, as inferred from local sequence alignments and from previously reported circular dichroism and resonance Raman spectra. These data afford structural information on a kind of [2Fe-2S] cluster-containing domain that occurs in a number of redox enzymes and complexes. In addition, together with previously published sequence alignments, they highlight the widespread distribution of the plant-type ferredoxin fold in bioenergetic systems encompassing anaerobic metabolism, photosynthesis, and aerobic respiratory chains.

Amino Acid Sequence↗

PubMed1994

Interleukin 4 increases type 5 acid phosphatase mRNA expression in murine bone marrow macrophages.

Type 5 acid phosphatase is a lysosomal enzyme expressed in cells of monocyte/macrophage lineage frequently used as a marker of osteoclastic differentiation. Oligonucleotide primers for DNA amplification were designed following sequence alignment of rat bone and human macrophage type 5 acid phosphatases. DNA (330 bp in length) obtained using these primers and reverse transcribed total cell RNA from in vitro generated murine osteoclastic cells was cloned and sequenced. DNA sequence analysis of two clones demonstrates that the amplified material was 91% and 96% identical to rat bone type 5 acid phosphatase at the nucleotide and amino acid level, respectively. Northern blots of murine tissue RNA show the presence of 1.5-kb transcripts that are most highly expressed in the long bones. Total cell RNA from the osteoclastic cells contain a marked level of type 5 acid phosphatase mRNA when compared to the levels seen in the tissue samples. Additionally, osteoclastic cell RNA contains two additional transcripts of 2.5 and 5 kb. Bone marrow macrophages grown in the presence of M-CSF express low levels of the 1.5-kb transcript with no signal observed for either of the two larger transcripts that were seen in the osteoclastic RNA samples. Importantly, bone marrow macrophage 1.5-kb type 5 acid phosphatase transcript levels are increased by interleukin 4 treatment in both a time and concentration-dependent manner. These findings indicate that type 5 acid phosphatase, while a cytochemical marker for osteoclasts, can be induced in macrophages by agents that block in vitro osteoclastic differentiation. Increased type 5 acid phosphatase may play a role in interleukin 4-stimulated monocyte activities.

Acid Phosphatase↗

PubMed2003

Molecular phylogenetic evidence refuting the hypothesis of Batoidea (rays and skates) as derived sharks.

Early morphological studies regarding the evolutionary history of elasmobranchs suggested sharks and batoids (skates and rays) were respectively monophyletic. More modern morphological cladistic studies, however, have tended to suggest that batoids are derived sharks, closely related to sawsharks and angelsharks, a phylogenetic arrangement known as the Hypnosqualea hypothesis. Very few molecular studies addressing interordinal relationships of elasmobranchs have been published; the few that do exist, are very limited in terms of both taxon representation and/or aligned sequence positions, and are insufficient to answer the question of whether batoids are derived sharks. The purpose of this study was to address this issue with more complete taxon representation, concomitant with a reasonable number of aligned sequence positions. The data set included a 2.4-kb segment of the mitochondrial 12S rRNA-tRNA valine-16S rRNA locus, and in terms of taxa, representatives of two orders of Batoidea, at least one representative of all orders of sharks, and as an outgroup, the widely recognized sister group to elasmobranchs-Holocephali. The results provide the first convincing molecular evidence for shark monophyly and the rejection of the Hypnosqualea hypothesis. Our phylogenetic placement of batoids as a basal elasmobranch lineage means that much of the current thinking regarding the evolution of morphological and life history characteristics in elasmobranchs needs to be re-evaluated.

Animals↗

PubMed1994

The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae.

Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.

Amino Acid Sequence↗

PubMed1992

Identification and characterization of foot-and-mouth disease virus O1 Burgwedel/1987 as an intertypic recombinant.

The foot-and-mouth disease virus field isolate Burgwedel/1987 subtype O1 was found to differ genetically from the antigenically related strain O1 Kaufbeuren within the region encoding the non-structural proteins. This genetic difference was indicated by the RNase mismatch cleavage method and confirmed by nucleotide sequencing. An alignment of sequences encoding proteinase 3C of the Burgwedel isolate and several other virus strains identified this isolate as an intertypic recombinant; the parent strains were O1 Kaufbeuren and a subtype C1 strain. Recombination occurred between nucleotide positions 5493 and 5521, within the region encoding peptide 3B1. Thus, the 5' three-quarters of the O1 genome were fused to the 3'-terminal quarter of the C1 genome. Other contemporary isolates from the same district are not recombinants. Sequence alignment distinguished four patterns of proteinase 3C-coding sequences among the virus strains analysed: subtypes A12, C1 and O1 exhibit one pattern each, and another pattern is common to subtypes A5, A10 and O2.

Aphthovirus↗

PubMed2002

Integrated gene and species phylogenies from unaligned whole genome protein sequences.

MOTIVATION: Most molecular phylogenies are based on sequence alignments. Consequently, they fail to account for modes of sequence evolution that involve frequent insertions or deletions. Here we present a method for generating accurate gene and species phylogenies from whole genome sequence that makes use of short character string matches not placed within explicit alignments. In this work, the singular value decomposition of a sparse tetrapeptide frequency matrix is used to represent the proteins of organisms uniquely and precisely as vectors in a high-dimensional space. Vectors of this kind can be used to calculate pairwise distance values based on the angle separating the vectors, and the resulting distance values can be used to generate phylogenetic trees. Protein trees so derived can be examined directly for homologous sequences. Alternatively, vectors defining each of the proteins within an organism can be summed to provide a vector representation of the organism, which is then used to generate species trees. RESULTS: Using a large mitochondrial genome dataset, we have produced species trees that are largely in agreement with previously published trees based on the analysis of identical datasets using different methods. These trees also agree well with currently accepted phylogenetic theory. In principle, our method could be used to compare much larger bacterial or nuclear genomes in full molecular detail, ultimately allowing accurate gene and species relationships to be derived from a comprehensive comparison of complete genomes. In contrast to phylogenetic methods based on alignments, sequences that evolve by relative insertion or deletion would tend to remain recognizably similar.

Algorithms↗

PubMed2006

Phytome: a platform for plant comparative genomics.

Phytome is an online comparative genomics resource that can be applied to functional plant genomics, molecular breeding and evolutionary studies. It contains predicted protein sequences, protein family assignments, multiple sequence alignments, phylogenies and functional annotations for proteins from a large, phylogenetically diverse set of plant taxa. Phytome serves as a glue between disparate plant gene databases both by identifying the evolutionary relationships among orthologous and paralogous protein sequences from different species and by enabling cross-references between different versions of the same gene curated independently by different database groups. The web interface enables sophisticated queries on lineage-specific patterns of gene/protein family proliferation and loss. This rich dataset is serving as a platform for the unification of sequence-anchored comparative maps across taxonomic families of plants. The Phytome web interface can be accessed at the following URL: http://www.phytome.org. Batch homology searches and bulk downloads are available upon free registration.

Databases, Genetic↗

PubMed2004

A secondary structural model of the 28S rRNA expansion segments D2 and D3 from rootworms and related leaf beetles (Coleoptera: Chrysomelidae; Galerucinae).

We analysed the secondary structure of two expansion segments (D2, D3) of the 28S rRNA gene from 229 leaf beetles (Coleoptera: Chrysomelidae), the majority of which are in the subfamily Galerucinae. The sequences were compared in a multiple sequence alignment, with secondary structure inferred primarily from the compensatory base changes in the conserved helices of the rRNA molecules. This comparative approach yielded thirty helices comprised of base pairs with positional covariation. Based on these leaf beetle sequences, we report an annotated secondary structural model for the D2 and D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related beetle taxa. This predicted structure, consisting of seven major compound helices, is mostly consistent with previously proposed models for the D2 and D3 expansion segments in insects. Despite a lack of conservation in the primary structure of these regions of insect 28S rRNA, the evolution of the secondary structure of these seven major motifs may be informative above the nucleotide level for higher-order phylogeny reconstruction of major insect lineages.

Animals↗

PubMed1997

Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish.

Earlier studies distinguished two classes of catfish light (L) chain (designated F and G). The cDNA structure and genomic organization of G L chain gene clusters has also been characterized previously. In this study, full length cDNA encoding F L chain was derived using PCR strategies based on the determined amino-terminal protein sequence. The encoded V region is readily delineated into framework regions (FR) and complementarity-determining regions (CDR). Multiple sequence alignments indicate that the F V(L) is closely related to kappa gene families. The F C(L) cannot be generally classified but it is structurally distinct from the C(L) regions of G: the amino acid sequence similarity is <35%. cDNA sequences representing processed sterile F transcripts of different loci were identified. Each sequence begins within the J(L) recombination signal sequence and extends downstream through the I(L)-C(L) segments. Genomic blots hybridized with C(L) probes indicate that there are at least 50 different C(L) segments. Based upon V(L) hybridization studies, different families of V(L) segments appear to be associated with closely related F C(L) segments. In characterized genomic clones, F gene segments are arranged in closely linked clusters with single copies of V(L), J(L), and C(L) segments within each cluster. The V(L) segments are located in opposite transcriptional polarity relative to the J(L) and C(L) segments, which indicates that V(L) segments rearrange by inversion. These combined studies establish that two structurally distinct classes of L chains are present in teleost fish and that both of the L chain classes evolved within a common organizational pattern of clustered segmental genes.

Amino Acid Sequence↗