PubMed Health⌕ Search

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 541 records · Page 30Linked to original sources
PubMed1995

Multiple alignment using hidden Markov models.

A simulated annealing method is described for training hidden Markov models and producing multiple sequence alignments from initially unaligned protein or DNA sequences. Simulated annealing in turn uses a dynamic programming algorithm for correctly sampling suboptimal multiple alignments according to their probability and a Boltzmann temperature factor. The quality of simulated annealing alignments is evaluated on structural alignments of ten different protein families, and compared to the performance of other HMM training methods and the ClustalW program. Simulated annealing is better able to find near-global optima in the multiple alignment probability landscape than the other tested HMM training methods. Neither ClustalW nor simulated annealing produce consistently better alignments compared to each other. Examination of the specific cases in which ClustalW outperforms simulated annealing, and vice versa, provides insight into the strengths and weaknesses of current hidden Markov model approaches.

Algorithms↗
PubMed1997

Specific detection of Serpulina hyodysenteriae and potentially pathogenic weakly beta-haemolytic porcine intestinal spirochetes by polymerase chain reaction targeting 23S rDNA.

A 2470-bp section of the 23S ribosomal DNA from Serpulina hyodysenteriae and five biochemically different groups of weakly beta-haemolytic porcine intestinal Serpulina strains was sequenced. The similarity between the sequenced strains was high (96.85% to 99.84%). A phylogenetic tree was estimated by the maximum likelihood method. The sequenced strains formed three groups. Serpulina hyodysenteriae and biochemical group II ('S. intermedius') formed a cluster, but 20 nucleotide positions were different between the two, suggesting that biochemical group II is a separate species. Another cluster consisted of the closely related biochemical group IIIa ('S. murdochii') and IIIb/c (S. innocens) (99.84% similarity), while biochemical group IV (S. pilosicoli) constituted a separate group with a relatively low similarity (96.85% to 97.01%) to the other groups. Three primer pairs were designed for specific PCR detection of the clinically important S. hyodysenteriae and biochemical group II and IV. PCR amplification was accomplished with DNA extracted from bacterial colonies by a simple boiling procedure, and with DNA extracted directly from porcine stool samples using a bead beating extraction procedure. The level of detection for the direct extraction and amplification method was 5 x 10(5) cells added g-1 normal faeces.

Animals↗
PubMed1996

The amino acid sequences, structure comparisons and inhibition kinetics of sheep cathepsin L and sheep stefin B.

Cathepsin L and stefin B were isolated from sheep liver, the cathepsin L being isolated by a low pH homogenisation method, which increases the proportion of the two-chain form of the enzyme, thus facilitating sequencing. The amino acid sequences of the isolated cathepsin L and stefin B were determined. The two-chain form of cathepsin L contains 217 amino acid residues and has an M(r) of 23,627. The sequence was obtained by sequencing the native active enzyme, the light and heavy chains and the peptides generated by cyanogen bromide cleavage. These peptides were aligned with peptides obtained by hydrolysis with endoproteinase Lys-C, glycyl endopeptidase and endoproteinase Glu-C. Sheep liver cathepsin L exhibits a high degree of sequence identity to human cathepsin L. Sheep stefin B consists of 98 amino acid residues and its calculated M(r) is 11,150. The inhibitor has its NH2-terminal amino acid residue blocked. Its amino acid sequence was determined by sequencing the peptides obtained by cleavage with cyanogen bromide and peptides obtained by hydrolysis with endoproteinase Glu-C and endoproteinase Lys-C. Sheep stefin B shows a high degree of sequence identity with bovine and human stefin B. The kinetics of the interaction between sheep cathepsin L and stefin B were determined, with the interaction of stefin B with papain used as a benchmark to compare with other published results. Despite the considerable homology between bovine and sheep stefin B, the kinetics of their interaction with papain and cathepsin L differed markedly, possibly due to the differences in the so-called "trunk" region of the cystatin molecule.

Amino Acid Sequence↗
PubMed2004

Scoring hidden Markov models to discriminate beta-barrel membrane proteins.

A new method is presented for identification of beta-barrel membrane proteins. It is based on a hidden Markov model (HMM) with an architecture obeying these proteins' construction principles. Once the HMM is trained, log-odds score relative to a null model is used to discriminate beta-barrel membrane proteins from other proteins. The method achieves only 10% false positive and false negative rates in a six-fold cross-validation procedure. The results compare favorably with existing methods. This method is proposed to be a valuable tool to quickly scan proteomes of entirely sequenced organisms for beta-barrel membrane proteins.

Algorithms↗
PubMed2004

Application of a new probabilistic model for recognizing complex patterns in glycans.

MOTIVATION: The study of carbohydrate sugar chains, or glycans, has been one of slow progress mainly due to the difficulty in establishing standard methods for analyzing their structures and biosynthesis. Glycans are generally tree structures that are more complex than linear DNA or protein sequences, and evidence shows that patterns in glycans may be present that spread across siblings and into further regions that are not limited by the edges in the actual tree structure itself. Current models were not able to capture such patterns. RESULTS: We have applied a new probabilistic model, called probabilistic sibling-dependent tree Markov model (PSTMM), which is able to inherently capture such complex patterns of glycans. Not only is the ability to capture such patterns important in itself, but this also implies that PSTMM is capable of performing multiple tree structure alignments efficiently. We prove through experimentation on actual glycan data that this new model is extremely useful for gaining insight into the hidden, complex patterns of glycans, which are so crucial for the development and functioning of higher level organisms. Furthermore, we also show that this model can be additionally utilized as an innovative approach to multiple tree alignment, which has not been applied to glycan chains before. This extension on the usage of PSTMM may be a major step forward for not only the structural analysis of glycans, but it may consequently prove useful for discovering clues into their function.

Algorithms↗
PubMed2004

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution.

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.

Animals↗
PubMed1997

Expression and upregulation of microtubule-associated protein 1B in cultured retinal pigment epithelial cells.

PURPOSE: During routine cell culture and under pathologic conditions, human retinal pigment epithelial (RPE) cells lose epithelial characteristics and change their morphology. In this study, changes in gene expression in RPE cells of different generations were evaluated by polymerase-chain-reaction-based differential display mRNA analysis (DD-RT-PCR). METHODS: Total RNA was prepared from freshly isolated and cultured human RPE cells of passages P0 and P3 and was subjected to DD-RT-PCR. One band with enhanced expression was excised, reamplified, and partially sequenced, using a modified dideoxy chain termination approach. Expression of the corresponding protein was ascertained by immunocytochemical analysis. RESULTS: Differential display RT-PCR showed enhanced expression of a specific RNA in P3 cells compared with that in P0 cells. Sequence alignment revealed 98% identity with the 3' end of the coding sequence of human microtubule-associated protein 1B (MAP1B). Confirmation of induced expression of MAP1B mRNA was obtained by PCR with specific primers and by immunocytochemical analysis in cultured RPE cells and in surgically removed epiretinal membranes from patients with proliferative vitreoretinopathy. No expression of MAP1B mRNA or protein was detected in freshly isolated RPE cells. CONCLUSIONS: Differential display RT-PCR in RPE cells with subsequent sequence analysis allows characterization of the maturation- and differentiation-dependent expression of previously undetected genes and gene products in cultured RPE cells.

Animals↗
PubMed2002

A comparison of profile hidden Markov model procedures for remote homology detection.

Profile hidden Markov models (HMMs) are amongst the most successful procedures for detecting remote homology between proteins. There are two popular profile HMM programs, HMMER and SAM. Little is known about their performance relative to each other and to the recently improved version of PSI-BLAST. Here we compare the two programs to each other and to non-HMM methods, to determine their relative performance and the features that are important for their success. The quality of the multiple sequence alignments used to build models was the most important factor affecting the overall performance of profile HMMs. The SAM T99 procedure is needed to produce high quality alignments automatically, and the lack of an equivalent component in HMMER makes it less complete as a package. Using the default options and parameters as would be expected of an inexpert user, it was found that from identical alignments SAM consistently produces better models than HMMER and that the relative performance of the model-scoring components varies. On average, HMMER was found to be between one and three times faster than SAM when searching databases larger than 2000 sequences, SAM being faster on smaller ones. Both methods were shown to have effective low complexity and repeat sequence masking using their null models, and the accuracy of their E-values was comparable. It was found that the SAM T99 iterative database search procedure performs better than the most recent version of PSI-BLAST, but that scoring of PSI-BLAST profiles is more than 30 times faster than scoring of SAM models.

Amino Acid Sequence↗
PubMed2002

Identification of sequence errors in HLA-DRB1*0801 and HLA-DRB1*12011.

We report the identification of previously unrecognised errors in the nucleotide sequences of two long established HLA-DRB1 alleles, DRB1*0801 and DRB1*12011. The errors were detected during development of sequencing based typing (SBT) methods for the HLA-DRB1 locus and were confirmed by sequencing cell lines from the 10th International Histocompatibility Workshop (IHW).

Alleles↗
PubMed2004

A method for cloning and sequencing long palindromic DNA junctions.

DNA sequences containing long adjacent inverted repeats (palindromes) are inherently unstable and are associated with many types of chromosomal rearrangements. The instability associated with palindromic sequences also creates difficulties in their molecular analysis: long palindromes (>250 bp/arm) are highly unstable in Escherichia coli, and cannot be directly PCR amplified or sequenced due to their propensity to form intra-strand hairpins. Here, we show that DNA molecules containing long palindromes (>900 bp/arm) can be transformed and stably maintained in Saccharomyces cerevisiae cells lacking a functional SAE2 gene. Treatment of the palindrome-containing DNA with sodium bisulfite at high temperature results in deamination of cytosine, converting it to uracil and thus reducing the propensity to form intra-strand hairpins. The bisulfite-treated DNA can then be PCR amplified, cloned and sequenced, allowing determination of the nucleotide sequence of the junctions. Our data demonstrates that long palindromes with either no spacer (perfect) or a 2 bp spacer can be stably maintained, recovered and sequenced from sae2Delta yeast cells. Since DNA sequences from mammalian cells can be gap repaired by their co-transformation into yeast cells with an appropriate vector, the methods described in this manuscript should provide some of the necessary tools to isolate and characterize palindromic junctions from mammalian cells.

Base Sequence↗
PubMed2006

Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae.

The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.

Amino Acid Sequence↗
PubMed1996

Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis.

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.

Base Sequence↗
PubMed2000

Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia.

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 10(4) CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the Chelex method could be used to detect fewer than 20 cultured Vibrio cells in sea-water or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.

Animals↗
PubMed2001

[Hepatitis C virus genotyping by restriction fragment length polymorphism of polymerase chain reaction products generated with a HCV detection kit].

Highly conserved sequence in the 5' untranslated region(UTR) of hepatitis C virus(HCV) genome have been targeted by most nucleic acid amplification-based detection assays, such as Amplicor HCV test, a commercially available assay kit. In this study, we classified HCV genotypes by direct sequencing determination for 5' UTR of nested-PCR after Amplicor HCV test. Then, based on the results of sequence, RFLP analysis after digestion of the nested PCR fragments with Hae III or Sau 3AI to classify HCV genotype was evaluated. RFLP analysis distinguished the type 1, 2a and 2b. Only one of 29 samples was not classified by RFLP analysis due to the point mutation of Hae III recognition site. HCV genotypes commonly found in JAPAN were classified into three types, 1b, 2a, and 2b. Also, RFLP analysis requires fewer resources than serotype grouping test. Hence, the present method provides an adaptable and rapid HCV genotyping in clinical laboratory in JAPAN.

Base Sequence↗
PubMed2004

Serological and genetic characterisation of a unique strain of adenovirus involved in an outbreak of epidemic keratoconjunctivitis.

AIMS: To characterise a novel strain of adenovirus (Ad) type Ad8 (genome type Ad8I) involved in an epidemic keratoconjunctivitis (EKC) outbreak in Hiroshima city using serological testing and sequence analysis of the fibre and hexon gene. METHODS: A neutralisation test (NT) was performed in microtitre plates containing a confluent monolayer of A549 cells using 100 tissue culture infectious doses of virus and type specific antisera. The haemagglutination inhibition test was also carried out in microtitre plates with rat erythrocytes using four haemagglutination units of virus and twofold dilutions of serum. The fibre gene was sequenced by generating overlapping polymerase chain reaction products or by direct sequencing of genomic DNA. Primer selection was based on alignment of the fibre genes of human adenovirus serotypes Ad8, Ad19, Ad37, Ad9, and Ad15 available from Gene Bank. RESULTS: The virus strain was specifically neutralised by anti-Ad8 antibodies, although there was a major crossreaction with anti-Ad9 antibodies. Haemagglutination was equally inhibited by anti-Ad8 and anti-Ad9 antibodies. The predicted amino acid sequences of the hypervariable regions (HVRs) of the Ad8I hexon gene showed higher homology with Ad9 (83.3%) than with Ad8 (62.0%). However, the Ad8I fibre knob was more homologous to Ad8 (94.4%) than to Ad9 (91.6%). CONCLUSIONS: Ad8I is a unique strain of adenovirus because of its lower genomic homology with Ad8, major crossreactivity with Ad9 in NT, and mixed genetic organisation of HVRs of the hexon gene. These factors may have enabled the virus to circumvent acquired immunity, resulting in the outbreak.

Adenovirus Infections, Human↗
PubMed1995

Sequence analysis of echoviruses in a major antigenic region eliciting enteroviral cross-reactive antibodies.

The amino acid sequence of an antigenic region known to elicit cross-reactive enteroviral IgG antibodies in VP1 is known for poliovirus and cox-sackievirus A and B. However, no corresponding data has been available for prevalent echovirus serotypes with great clinical impact. Such information was obtained by amplification of this region of the echovirus genome by PCR using biotinylated primers. The amplicon was subjected to solid phase sequencing using the dideoxy chain-termination method. Translated amino acid sequences for residues 26-55 of VP1 of the echoviruses revealed that the known cross-reactive region is highly conserved also in the echovirus serotypes.

Amino Acid Sequence↗
PubMed2005

Using concept mapping to design an indicator framework for addiction treatment centres.

OBJECTIVE: The objective of this study is to determine an indicator framework for addiction treatment centres based on the demands of stakeholders and in alignment with the European Foundation for Quality Management (EFQM) Excellence Model. SETTING: The setting is the Jellinek Centre based in Amsterdam, the Netherlands, which serves as a prototype for an addiction treatment centre. METHOD: Concept mapping was used in the construction of the indicator framework. During the 1-day workshop, 16 stakeholders generated, prioritized and sorted 73 items concerning quality and performance. Multidimensional scaling and cluster analysis was applied in constructing a framework consisting of two dimensions and eight clusters. RESULTS: The horizontal axis of the indicator framework is named 'Organization' and has two poles, namely, 'Processes' and 'Results'. The vertical axis is named ' Task' and the poles are named 'Efficient treatment' and 'Prevention programs'. The eight clusters in the two-dimensional framework are arranged in the following, prioritized sequence: 'Efficient treatment network', 'Effective service', ' Target group', 'Quality of life', 'Efficient service', 'Knowledge transfer', 'Reducing addiction related problems', and 'Prevention programs'. The most important items in the framework are: 'patients are satisfied with their treatment', 'early interventions', and 'efficient treatment chain'. CONCLUSION: The indicator framework aligns with three clusters of the results criteria of the EFQM Excellence Model. It is based on the stakeholders' perspectives and is believed to be specific for addiction treatment centres. The study demonstrates that concept mapping is a suitable strategy for generating indicator frameworks.

Benchmarking↗