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At least 955 records · Page 53Linked to original sources

PubMed2001

Genetic variation in the 3' non-coding region of dengue viruses.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

3' Untranslated Regions↗

PubMed1997

Evolution of transcription-regulating proteins: caveat lector!

The paper of Hawkins et al. [Gene 146 (1994) 145-158] reports incorrect descriptions of mutant phenotypes, omits mention of the absence of a highly relevant glutamine-binding site and contains sequence alignments which might mislead the reader. Extensive sequence analysis reveals as untenable a central hypothesis of the paper concerning a possible evolutionary relationship between anthranilate synthetases and the transcription factors mediating nitrogen metabolite repression in fungi.

Anthranilate Synthase↗

PubMed2001

Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: identification of a myotubularin-related protein lacking catalytic activity.

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

Amino Acid Sequence↗

PubMed2004

Cloning, characterization and prokaryotic expression of cytosolic malate dehydrogenase from Oryza sativa.

cDNA sequences of malate dehydrogenase (MDH) were cloned from various species, and MDH was identified to play an important role in cell energy metabolism. Here, we present the isolation and characterization of its homologue (OscMDH) in Oryza sativa. Comparison of the results to the genome details indicated that OscMDH consisted of seven exons. Sequence alignment showed that the deduced amino acid sequence of OscMDH shared a significant similarity with cMDH protein in Zea mays, as well as with other cMDH gene products. The different expression patterns of OscMDH in different tissues revealed that OscMDH mRNA was highly transcribed in either young panicle or immature seed, while its abundance was much low in green leaf and root. A nearly 56-kDa fusion protein generated by adding a Trx-His-tag at the N-terminal of OscMDH was induced by IPTG in Escherichia coli BL21 and an obvious MDH activity was detected in the protein by native PAGE analysis. All these results suggest that OscMDH encodes a cytosolic MDH in rice.

Actins↗

PubMed2004

The ERPIN server: an interface to profile-based RNA motif identification.

ERPIN is an RNA motif identification program that takes an RNA sequence alignment as an input and identifies related sequences using a profile-based dynamic programming algorithm. ERPIN differs from other RNA motif search programs in its ability to capture subtle biases in the training set and produce highly specific and sensitive searches, while keeping CPU requirements at a practical level. In its latest version, ERPIN also computes E-values, which tell biologists how likely they are to encounter a specific sequence match by chance-a useful indication of biological significance. We present here the ERPIN online search interface (http://tagc.univ-mrs.fr/erpin/). This web server automatically performs ERPIN searches for different RNA genes or motifs, using predefined training sets and search parameters. With a couple of clicks, users can analyze an entire bacterial genome or a genomic segment of up to 5Mb for the presence of tRNAs, 5S rRNAs, SRP RNA, C/D box snoRNAs, hammerhead motifs, miRNAs and other motifs. Search results are displayed with sequence, score, position, E-value and secondary structure graphics. An example of a complete genome scan is provided, as well as an evaluation of run times and specificity/sensitivity information for all available motifs.

Internet↗

PubMed2005

Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A.

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99.5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

Animals↗

PubMed1999

CAP3: A DNA sequence assembly program.

We describe the third generation of the CAP sequence assembly program. The CAP3 program includes a number of improvements and new features. The program has a capability to clip 5' and 3' low-quality regions of reads. It uses base quality values in computation of overlaps between reads, construction of multiple sequence alignments of reads, and generation of consensus sequences. The program also uses forward-reverse constraints to correct assembly errors and link contigs. Results of CAP3 on four BAC data sets are presented. The performance of CAP3 was compared with that of PHRAP on a number of BAC data sets. PHRAP often produces longer contigs than CAP3 whereas CAP3 often produces fewer errors in consensus sequences than PHRAP. It is easier to construct scaffolds with CAP3 than with PHRAP on low-pass data with forward-reverse constraints.

Algorithms↗

PubMed1995

Analysis of Neisseria meningitidis class 3 outer membrane protein gene variable regions and type identification using genetic techniques.

The class 3 porin proteins of Neisseria meningitidis stimulate bactericidal antibodies and express serotype-specific antigenic epitopes. Sequence analysis of porB genes for the class 3 proteins revealed regions of variability that map to surface-exposed loops. To evaluate the relationship between serotype and variable-region (VR) genotype, sequences from the 11 class 3-expressing serotype strains and 3 additional serotype 4 strains were analyzed by molecular techniques. Multiple-sequence alignment revealed a limited number of unique sequences at each of four VRs (VR1 to VR4), ranging from four unique sequences at VR1 to seven sequence patterns at VR2 and VR4. Serotype-specific VR sequences were found in each of the four VRs, suggesting that each VR has immunologic importance. Five serotypes had at least one VR sequence that was unique. Three serotypes which had sequences in common with other serotypes at each VR were distinguished by examining multiple VRs. Serotype 3 was identical to serotype 19 at each VR, and serotype 8 was identical to serotype 18 at each VR. Serotypes 4 and 21 were identical at VR1 and significantly different at VR3 and VR4. A subpopulation of serotype 4 strains with a unique VR2 sequence was identified. The serotypes which were grouped with closely related or identical sequences at one VR were grouped with different serotypes at other VRs consistent with the pattern of genetic mosaicism described for the porA (class 1 protein) gene. Hybridization assays demonstrated the ability to identify VR genotypes and distinguish serotypes using biotin-labelled oligonucleotide probes. This information may be useful in strain selection for vaccine development, in epidemiologic studies to determine the prevalence of the individual VR genotype (especially among nonserotypeable strains) and, combined with PCR, in the identification of culture-negative suspected meningococcal cases.

Amino Acid Sequence↗

PubMed2006

Cloning and expression of a mannose-binding jacalin-related lectin from leaves of Japanese cycad (Cycas revoluta Thunb.).

Cycad leaf lectin (CRLL), a mannose-recognizing jacalin-related lectin (mJRL), was first cloned as a gymnosperm lectin and expressed. The cDNA sequence of CRLL (DDBJ, accession no. AB198328), coding 291 amino acid residues, has a tandem repeat of about 150 amino acids divided into N- and C-terminal domains as Japanese chestnut mJRL. Sequence alignment showed deletion and insertion of the sequence, and its putative carbohydrate-binding sites showed some differences from other JRLs. PCR analysis showed that this lectin was expressed in the cycad leaf but not in the root or seed. Recombinant CRLL (rCRLL) was expressed in Escherichia coli and purified by affinity chromatography after refolding procedures. Properties of active rCRLL appeared to be almost the same as those of native CRLL.

Amino Acid Sequence↗

PubMed2000

Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza.

Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H(+)-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.

Amino Acid Sequence↗

PubMed2006

Logarithmic gap costs decrease alignment accuracy.

BACKGROUND: Studies on the distribution of indel sizes have consistently found that they obey a power law. This finding has lead several scientists to propose that logarithmic gap costs, G (k) = a + c ln k, are more biologically realistic than affine gap costs, G (k) = a + bk, for sequence alignment. Since quick and efficient affine costs are currently the most popular way to globally align sequences, the goal of this paper is to determine whether logarithmic gap costs improve alignment accuracy significantly enough the merit their use over the faster affine gap costs. RESULTS: A group of simulated sequences pairs were globally aligned using affine, logarithmic, and log-affine gap costs. Alignment accuracy was calculated by comparing resulting alignments to actual alignments of the sequence pairs. Gap costs were then compared based on average alignment accuracy. Log-affine gap costs had the best accuracy, followed closely by affine gap costs, while logarithmic gap costs performed poorly. Subsequently a model was developed to explain the results. CONCLUSION: In contrast to initial expectations, logarithmic gap costs produce poor alignments and are actually not implied by the power-law behavior of gap sizes, given typical match and mismatch costs. Furthermore, affine gap costs not only produce accurate alignments but are also good approximations to biologically realistic gap costs. This work provides added confidence for the biological relevance of existing alignment algorithms.

Algorithms↗

PubMed1996

Constructing amino acid residue substitution classes maximally indicative of local protein structure.

Using an information theoretic formalism, we optimize classes of amino acid substitution to be maximally indicative of local protein structure. Our statistically-derived classes are loosely identifiable with the heuristic constructions found in previously published work. However, while these other methods provide a more rigid idealization of physicochemically constrained residue substitution, our classes provide substantially more structural information with many fewer parameters. Moreover, these substitution classes are consistent with the paradigmatic view of the sequence-to-structure relationship in globular proteins which holds that the three-dimensional architecture is predominantly determined by the arrangement of hydrophobic and polar side chains with weak constraints on the actual amino acid identities. More specific constraints are imposed on the placement of prolines, glycines, and the charged residues. These substitution classes have been used in highly accurate predictions of residue solvent accessibility. They could also be used in the identification of homologous proteins, the construction and refinement of multiple sequence alignments, and as a means of condensing and codifying the information in multiple sequence alignments for secondary structure prediction and tertiary fold recognition.

Amino Acid Sequence↗

PubMed2003

Cloning and characterisation of the first drug-metabolising canine UDP-glucuronosyltransferase of the 2B subfamily.

Glucuronidation is a major route of clearance for a diverse set of both drug and endogenous substrates. The present study was undertaken to redress the lack of molecular information currently available on drug glucuronidation by the dog, a species widely used in metabolism studies by the pharmaceutical industry. A novel dog uridine diphosphate glucuronosyltransferase (UGT), designated UGT2B31 (GenBank Accession Number: AY135176), has been isolated from a dog cDNA library, expressed in V79 cells and characterised using various methods: (i) UGT2B31 sequence has been compared with mammalian UGT sequences using both sequence alignments and phylogenetic analysis; and (ii) the substrate specificity of UGT2B31 has been determined using functional analysis and compared with that obtained using UGT2B7 and dog liver microsomes. The following results were obtained: (i) sequence alignments between UGT2B31 and UGT2B15 gave the greatest degree of identity (76%); however, human UGT2B4, human UGT2B7, monkey UGT2B9 (all 75%), and rat UGT2B1 (73%) also gave a high degree of identity; (ii) phylogenetic analysis determined UGT2B31 to be most closely related to rat UGT2B1; (iii) UGT2B31 displayed a substrate specificity similar to human UGT2B7 and rat UGT2B1, catalysing the glucuronidation of phenols, opioids, and carboxylic acid-containing drugs; and (iv) UGT2B31 only formed morphine-3-glucuronide; however, kinetic analysis determined the K(m) of this reaction to be similar to that observed with UGT2B7 (both approximately 1300 microM). The results suggest that UGT2B31 plays a crucial role in drug detoxification by the dog and may be the canine equivalent of human UGT2B7.

Amino Acid Sequence↗

PubMed2002

SUPFAM--a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes.

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying 'priority proteins' for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.

Animals↗

PubMed1996

XFINGER: a tool for searching and visualising protein fingerprints and patterns.

A tool for searching pattern and fingerprint databases is described. Fingerprints are groups of motifs excised from conserved regions of sequence alignments and used for iterative database scanning. The constituent motifs are thus encoded as small alignments in which sequence information is maximised with each database pass; they therefore differ from regular-expression patterns, in which alignments are reduced to single consensus sequences. Different database formats have evolved to store these disparate types of information, namely the PROSITE dictionary of patterns and the PRINTS fingerprint database, but programs have not been available with the flexibility to search them both. We have developed a facility to do this: the system allows query sequences to be scanned against either PROSITE, the full PRINTS database, or against individual fingerprints. The results of fingerprint searches are displayed simultaneously in both text and graphical windows to render them more tangible to the user. Where structural coordinates are available, identified motifs may be visualised in a 3D context. The program runs on Silicon Graphics machines using GL graphics libraries and on machines with X servers supporting the PEX extension: its use is illustrated here by depicting the location of low-density lipoprotein-binding (LDL) motifs and leucine-rich repeats in a mosaic G-protein-coupled receptor (GPCR).

Amino Acid Sequence↗

PubMed2006

Sequence comparison by sequence harmony identifies subtype-specific functional sites.

Multiple sequence alignments are often used to reveal functionally important residues within a protein family. They can be particularly useful for the identification of key residues that determine functional differences between protein subfamilies. We present a new entropy-based method, Sequence Harmony (SH) that accurately detects subfamily-specific positions from a multiple sequence alignment. The SH algorithm implements a novel formula, able to score compositional differences between subfamilies, without imposing conservation, in a simple manner on an intuitive scale. We compare our method with the most important published methods, i.e. AMAS, TreeDet and SDP-pred, using three well-studied protein families: the receptor-binding domain (MH2) of the Smad family of transcription factors, the Ras-superfamily of small GTPases and the MIP-family of integral membrane transporters. We demonstrate that SH accurately selects known functional sites with higher coverage than the other methods for these test-cases. This shows that compositional differences between protein subfamilies provide sufficient basis for identification of functional sites. In addition, SH selects a number of sites of unknown function that could be interesting candidates for further experimental investigation.

Algorithms↗

PubMed1997

Statistical significance of hierarchical multi-body potentials based on Delaunay tessellation and their application in sequence-structure alignment.

Statistical potentials based on pairwise interactions between C alpha atoms are commonly used in protein threading/fold-recognition attempts. Inclusion of higher order interaction is a possible means of improving the specificity of these potentials. Delaunay tessellation of the C alpha-atom representation of protein structure has been suggested as a means of defining multi-body interactions. A large number of parameters are required to define all four-body interactions of 20 amino acid types (20(4) = 160,000). Assuming that residue order within a four-body contact is irrelevant reduces this to a manageable 8,855 parameters, using a nonredundant dataset of 608 protein structures. Three lines of evidence support the significance and utility of the four-body potential for sequence-structure matching. First, compared to the four-body model, all lower-order interaction models (three-body, two-body, one-body) are found statistically inadequate to explain the frequency distribution of residue contacts. Second, coherent patterns of interaction are seen in a graphic presentation of the four-body potential. Many patterns have plausible biophysical explanations and are consistent across sets of residues sharing certain properties (e.g., size, hydrophobicity, or charge). Third, the utility of the multi-body potential is tested on a test set of 12 same-length pairs of proteins of known structure for two protocols: Sequence-recognizes-structure, where a query sequence is threaded (without gap) through the native and a non-native structure; and structure-recognizes-sequence, where a query structure is threaded by its native and another non-native sequence. Using cross-validated training, protein sequences correctly recognized their native structure in all 24 cases. Conversely, structures recognized the native sequence in 23 of 24 cases. Further, the score differences between correct and decoy structures increased significantly using the three- or four-body potential compared to potentials of lower order.

Models, Chemical↗

PubMed2006

Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes.

Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.

Amino Acid Sequence↗