SEARCH · PubMed Health

Results for “alignment chaining method”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 973 records · Page 54Linked to original sources

PubMed1995

A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk.

A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.

Animals↗

PubMed2004

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood.

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).

Animals↗

PubMed2000

High prevalence of GB virus C/hepatitis G virus in Kinshasa, Democratic Republic of Congo: a phylogenetic analysis.

A prevalence of 10.3% of GB virus C (GBV-C)/hepatitis G virus (HGV) carriers was found in 97 pregnant women from Kinshasa, Congo (formerly Zaire), while prevalences of 1%, 4.1%, and 0% were found for hepatitis C virus, human immunodeficiency virus, and human T-lymphotropic virus respectively. Phylogenetic analysis of the ten GBV-C/HGV positives based on the 5' non-coding region using three different methods identified consistently three GBV-C/HGV genotypes. Four main clades were found within the type 1 sequences. All the Congolese isolates are GBV-C/HGV type 1 in two different clades. The clustering of seven Congolese isolates was inconsistent in different methods. Further likelihood-mapping analysis showed a well-resolved phylogeny, confirming the clustering of the seven Congolese isolates with a Belgian strain representing a new clade in the GBV-C/HGV type 1 sequences.

Deltaretrovirus Infections↗

PubMed2001

Detection of conserved physico-chemical characteristics of proteins by analyzing clusters of positions with co-ordinated substitutions.

MOTIVATION: It is known that the physico-chemical characteristics of proteins underlying specific folding of the polypeptide chain and the protein function are evolutionary conserved. Detection of such characteristics while analyzing homologous sequences would expand essentially the knowledge on protein function, structure, and evolution. These characteristics are maintained constant, in particular, by co-ordinated substitutions. In this process, the destabilizing effect of a substitution may be compensated by another substitution at a different position within the same protein, making the overall change in this protein characteristic insignificant. Consequently, the patterns of co-ordinated substitutions contain important information on conserved physico-chemical properties of proteins, requiring their investigation and development of the corresponding methods and software for correlation analysis of protein sequences available to a wide range of users. RESULTS: A software package for analyzing correlated amino acid substitutions at different positions within aligned protein sequences was developed. The approach implies searching for evolutionary conserved physico-chemical characteristics of proteins based on the information on the pairwise correlations of amino acid substitutions at different protein positions. The software was applied to analyze DNA-binding domains of the homeodomain class. As a result, two conservative physico-chemical characteristics preserved due to the co-ordinated substitutions at certain groups of positions in the protein sequence. Possible functional roles of these characteristics are discussed. AVAILABILITY: The program package is available at http://wwwmgs.bionet.nsc.ru/programs/CRASP/.

Algorithms↗

PubMed1991

Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures.

In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.

Amino Acid Sequence↗

PubMed2006

Evolutionary coupling of structural and functional sequence information in the intracellular lipid-binding protein family.

We have mined the evolutionary record for the large family of intracellular lipid-binding proteins (iLBPs) by calculating the statistical coupling of residue variations in a multiple sequence alignment using methods developed by Ranganathan and coworkers (Lockless and Ranganathan, Science 1999:286;295-299). The 213 sequences analyzed have a wide range of ligand-binding functions as well as highly divergent phylogenetic origins, assuring broad sampling of sequence space. Emerging from this analysis were two major clusters of coupled residues, which when mapped onto the structure of a representative iLBP under study in our laboratory, cellular retinoic-acid binding protein I, are largely contiguous and provide useful points of comparison to available data for the folding of this protein. One cluster comprises a predominantly hydrophobic core away from the ligand-binding site and likely represents key structural information for the iLBP fold. The other cluster includes the portal region where ligand enters its binding site, regions of the ligand-binding cavity, and the region where the 10-stranded beta-barrel characteristic of this family closes (between strands 1' and 10). Linkages between these two clusters suggest that evolutionary pressures on this family constrain structural and functional sequence information in an interdependent fashion. The necessity of the structure to wrap around a hydrophobic ligand confounds the typical sequestration of hydrophobic side chains. Additionally, ligand entry and exit require these structures to have a capacity for specific conformational change during binding and release. We conclude that an essential and structurally apparent separation of local and global sequence information is conserved throughout the iLBP family.

Amino Acid Sequence↗

PubMed2002

Identification of randomly selected colonies of lactobacilli from normal vaginal fluid by pyrosequencing of the 16S rDNA variable V1 and V3 regions.

The present study aimed to characterize lactobacilli in vaginal fluid from 23 adult healthy women by using high-throughput DNA sequencing for identification of a large number of randomly selected colonies appearing on Rogosa and blood agar. The typing method was based on broad-range PCR of 16S rRNA gene variable regions V1 and V3, pyrosequencing, and classification of the fragments by alignment with NCBI-catalogued sequences and type strain sequences. Four major groups of sequences were found among the 402 isolates clearly corresponding to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii when compared to the sequences obtained for type strains. Our results indicate that pyrosequencing of 16S rRNA gene fragments as used here is a fast and reliable method well suited for identification to the species level, even within the Lactobacillus acidophilus complex.

Adult↗

PubMed2003

Comparison of glycoprotein B (gB) variants of the elephant endotheliotropic herpesvirus (EEHV) isolated from Asian elephants (Elephas maximus).

The recently described elephant endotheliotropic herpesviruses (EEHV) have been associated with the deaths of numerous captive elephants. A proposed tool for the detection of EEHV infection in elephants is the PCR-based screening for EEHV-DNA in whole blood samples. Unfortunately, this detection method has only been successful in post-mortem analyses or in animals already displaying clinical signs of EEHV disease, thus rendering this method unsuitable for identification of carrier elephants. Here, we focus on glycoprotein B (gB) for serologic assay development, since gB is an envelope protein known to induce a neutralising antibody response in other herpesvirus infections. We sequenced the entire gB gene from five Asian elephants with EEHV, representing four different gB variants. Computer-aided methods were used to predict functionally important regions within EEHVgB. An extra-cytoplasmic region of 153 amino acids was predicted to be under positive selection and may potentially contain antigenic determinants that will be useful for future serologic assay development.

Amino Acid Sequence↗

PubMed2005

Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information.

In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix-turn-helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of 'partial' domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function.

Amino Acid Sequence↗

PubMed2003

DDBASE2.0: updated domain database with improved identification of structural domains.

MOTIVATION: Although many methods are available for the identification of structural domains from protein three-dimensional structures, accurate definition of protein domains and the curation of such data for a large number of proteins are often possible only after manual intervention. The availability of domain definitions for protein structural entries is useful for the sequence analysis of aligned domains, structure comparison, fold recognition procedures and understanding protein folding, domain stability and flexibility. RESULTS: We have improved our method of domain identification starting from the concept of clustering secondary structural elements, but with an intention of reducing the number of discontinuous segments in identified domains. The results of our modified and automatic approach have been compared with the domain definitions from other databases. On a test data set of 55 proteins, this method acquires high agreement (88%) in the number of domains with the crystallographers' definition and resources such as SCOP, CATH, DALI, 3Dee and PDP databases. This method also obtains 98% overlap score with the other resources in the definition of domain boundaries of the 55 proteins. We have examined the domain arrangements of 4592 non-redundant protein chains using the improved method to include 5409 domains leading to an update of the structural domain database. AVAILABILITY: The latest version of the domain database and online domain identification methods are available from http://www.ncbs.res.in/~faculty/mini/ddbase/ddbase.html SUPPLEMENTARY INFORMATION: http://www.ncbs.res.in/~faculty/mini/ddbase/supplementary/supplementary.html

Amino Acid Sequence↗

PubMed2002

The use of structure information to increase alignment accuracy does not aid homologue detection with profile HMMs.

MOTIVATION: The best quality multiple sequence alignments are generally considered to derive from structural superposition. However, no previous work has studied the relative performance of profile hidden Markov models (HMMs) derived from such alignments. Therefore several alignment methods have been used to generate multiple sequence alignments from 348 structurally aligned families in the HOMSTRAD database. The performance of profile HMMs derived from the structural and sequence-based alignments has been assessed for homologue detection. RESULTS: The best alignment methods studied here correctly align nearly 80% of residues with respect to structure alignments. Alignment quality and model sensitivity are found to be dependent on average number, length, and identity of sequences in the alignment. The striking conclusion is that, although structural data may improve the quality of multiple sequence alignments, this does not add to the ability of the derived profile HMMs to find sequence homologues. SUPPLEMENTARY INFORMATION: A list of HOMSTRAD families used in this study and the corresponding Pfam families is available at http://www.sanger.ac.uk/Users/sgj/alignments/map.html CONTACT: sgj@sanger.ac.uk

Amino Acid Sequence↗

PubMed2006

Hidden Markov multiple event sequence models: A paradigm for the spatio-temporal analysis of fMRI data.

This paper presents a novel, completely unsupervised fMRI brain mapping method that addresses the three problems of hemodynamic response function (HRF) variability, hemodynamic event timing, and fMRI response non-linearity. Spatial and temporal information are directly taken into account into the core of the activation detection process. In practice, activation detection at voxel v is formulated in terms of temporal alignment between sequences of hemodynamic response onsets (HROs) detected in the fMRI signal at v and in the spatial neighborhood of v, and the input sequence of stimuli or stimulus onsets. Event-related and epoch paradigms are considered. The multiple event sequence alignment problem is solved within the probabilistic framework of hidden Markov multiple event sequence models (HMMESMs), a new class of hidden Markov models. Results obtained on real and synthetic data significantly outperform those obtained with the popular statistical parametric mapping (SPM2) method without requiring any prior definition of the expected activation patterns, the HMMESM mapping approach being completely unsupervised.

Adolescent↗

PubMed2003

[Clinical detection of polymerase gene of SARS-associated coronavirus].

OBJECTIVE: To analyze the heterogeneity of polymerase gene fragment of SARS-associated coronavirus from SARS patients, and establish a RT-PCR method for detecting SARS-associated coronavirus. METHODS: RT-PCR was performed using SARS coronavirus-specific primers to amplify the polymerase gene fragment of SARS-associated coronavirus from specimens of suspected and established SARS cases. The amplicons were cloned and sequenced. All the obtained sequences were compared with the sequence of published SARS-associated coronavirus, and alignment was proceeded with other coronavirus sequences. RESULTS: Specific amplicons can be amplified from the sputum samples, throat swab and plasma of most SARS patients, and 8 were random selected and sequenced. All of them possessed 100% homology with the published SARS-associated coronavirus sequence, while all the negative controls were RT-PCR negative. Nucleotide-sequence and amino acid-sequence alignment of the fragment BNI109 with other six known coronavirus show that the fragment BNI109 is more close to bovine coronavirus(BCV) and murine hepatitis virus(MHV). The BNI109 fragment showed 75% homology with BCV and MHV at amino acid level. CONCLUSION: The polymerase fragment BNI109 of SARS coronavirus is highly conservative and is suitable for detecting SARS-associated coronavirus using RT-PCR method.

Amino Acid Sequence↗

PubMed2002

A comparative analysis of PCR-based detection methods for avian malaria.

Here, 4 polymerase chain reaction (PCR) assays are compared to test for the presence of avian malaria, including both the Plasmodium and Haemoproteus genera, in 29 different species of African rainforest birds. Two of these PCR assays use primer sets that amplify fragments of the cytochrome b (cyt b) gene of Plasmodium; the other 2 target the 18S ribosomal subunit gene. These PCR assays were performed using genomic DNA extracted from blood and subsequently compared with the results obtained by microscopic examination of blood smears taken from the same individuals. The 2 primer sets amplifying the cyt b gene were found to perform more reliably than those that target the 18S rRNA gene and yielded a substantial number of positive samples that were undetected by blood smear analysis. Of all the individuals screened by PCR, 40% tested positive for avian malaria, whereas 27% tested positive by blood smear analysis. Although sequence variation in the parasites may prohibit the specific alignment of primers and the subsequent PCR amplification of some individuals, PCR, once optimized, is faster, cheaper, and more reliable than blood smear analysis for large-scale screening.

Animals↗

PubMed2006

A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles.

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChalphaE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.

Alleles↗

PubMed2002

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples.

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was < or = ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle 'one step' NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a 'one step' RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.

Base Sequence↗

PubMed1997

Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

BACKGROUND: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. RESULTS: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. CONCLUSIONS: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Amino Acid Sequence↗

PubMed2005

Calibrating E-values for hidden Markov models using reverse-sequence null models.

MOTIVATION: Hidden Markov models (HMMs) calculate the probability that a sequence was generated by a given model. Log-odds scoring provides a context for evaluating this probability, by considering it in relation to a null hypothesis. We have found that using a reverse-sequence null model effectively removes biases owing to sequence length and composition and reduces the number of false positives in a database search. Any scoring system is an arbitrary measure of the quality of database matches. Significance estimates of scores are essential, because they eliminate model- and method-dependent scaling factors, and because they quantify the importance of each match. Accurate computation of the significance of reverse-sequence null model scores presents a problem, because the scores do not fit the extreme-value (Gumbel) distribution commonly used to estimate HMM scores' significance. RESULTS: To get a better estimate of the significance of reverse-sequence null model scores, we derive a theoretical distribution based on the assumption of a Gumbel distribution for raw HMM scores and compare estimates based on this and other distribution families. We derive estimation methods for the parameters of the distributions based on maximum likelihood and on moment matching (least-squares fit for Student's t-distribution). We evaluate the modeled distributions of scores, based on how well they fit the tail of the observed distribution for data not used in the fitting and on the effects of the improved E-values on our HMM-based fold-recognition methods. The theoretical distribution provides some improvement in fitting the tail and in providing fewer false positives in the fold-recognition test. An ad hoc distribution based on assuming a stretched exponential tail does an even better job. The use of Student's t to model the distribution fits well in the middle of the distribution, but provides too heavy a tail. The moment-matching methods fit the tails better than maximum-likelihood methods. AVAILABILITY: Information on obtaining the SAM program suite (free for academic use), as well as a server interface, is available at http://www.soe.ucsc.edu/research/compbio/sam.html and the open-source random sequence generator with varying compositional biases is available at http://www.soe.ucsc.edu/research/compbio/gen_sequence

Algorithms↗