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 901 records · Page 50Linked to original sources

PubMed1998

Cloning and characterisation of the Proteus mirabilis xerD gene.

The Xer site-specific recombination system is involved in the stable maintenance of replicons (certain plasmids and chromosomes) in Escherichia coli and other bacteria by converting multimers into monomers. This system requires a cis-acting DNA sequence (the chromosomal dif site or the ColE1 cer site) and two trans-acting factors: the XerC and XerD recombinases, which belong to the lambda integrase family of tyrosine site-specific recombinases. In addition, in order to resolve plasmid multimers into monomers, two additional factors are required: the ArgR and PepA proteins. We have previously shown the presence of xerC and xerD genes (and their function) by Southern hybridisation and by in vivo recombination in a wide variety of Enterobacteriaceac. We have now cloned and sequenced the xerD gene of Proteus mirabilis using degenerate and inverse PCR methods. This gene encodes a tyrosine recombinase which is highly similar to the E. coli XerD recombinase, is capable of complementing an E. coli xerD mutant, and displays sequence-specific DNA binding activity.

Amino Acid Sequence↗

PubMed1999

The effects of ordered-series-of-motifs anchoring and sub-class modeling on the generation of HMMs representing highly divergent protein sequences.

Hidden Markov Models (HMMs) provide a flexible method for representing protein sequence data. Highly divergent data require a more complex approach to HMM generation than previously demonstrated. We describe a strategy of motif anchoring and sub-class modeling that aids in the construction of more informative HMMs as determined by a new algorithm called a stability measure.

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↗

PubMed1994

Genetic evidence for a hantavirus enzootic in deer mice (Peromyscus maniculatus) captured a decade before the recognition of hantavirus pulmonary syndrome.

To determine if the emergence of hantavirus pulmonary syndrome in the United States was a consequence of recent amino-acid altering mutations and/or genetic reassortment with pathogenic hantaviruses, we examined lung and spleen tissues from seropositive deer mice trapped in August 1983 in Mono County, California, for hantaviral RNA by reverse transcriptase-directed polymerase chain reaction. Alignment and comparison of 1485 nucleotides of the S and M genomic segments enzymatically amplified from these tissues indicated that these deer mice harbored a hantavirus which was genetically similar, differing by less than 2% at the deduced amino-acid level, to the hantavirus implicated in cases of hantavirus pulmonary syndrome occurring in the Four-Corners region of New Mexico, Arizona, Utah, and Colorado in 1993. The peromyscine rodent-borne hantaviruses were, in turn, genetically distinct from other well-characterized hantaviruses, diverging by approximately 30% from Prospect Hill and Puumala viruses at the nucleotide and amino acid levels. Phylogenetic analysis using the maximum parsimony, neighbor-joining, and unweighted pair-group methods indicated that the Peromyscus-derived hantavirus shared a common ancestry with arvicolid rodent-borne hantaviruses. Overall congruency of the phylogenetic trees based on the S and M genomic sequences supported the evolutionary position of the peromyscine rodent-borne hantaviruses. Our data also establish the existence of a hantavirus enzootic in deer mice long before the recognition of hantavirus pulmonary syndrome in the United States.

Amino Acid Sequence↗

PubMed1988

Amino acid substitutions in structurally related proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix.

Amino acid substitutions in evolutionarily related proteins have been studied from a structural point of view. We consider here that an amino acid al in a protein p1 has been replaced by the amino acid a2 in the structurally similar protein p2 if, after superposition of the p1 and p2 structures, the a1 and a2 C alpha atoms are no more than 1.2 A apart. Thirty-two proteins, grouped in 11 classes, have been analysed by this method. This produced 2860 amino acid pairs (substitutions), which were analysed by multi-dimensional statistical methods. The main results are as follows: (1) according to the observed exchangeability of amino acid side-chains, only four groups (strong clusters) could be delineated; (i) Ile and Val, (ii) Leu and Met, (iii) Lys, Arg and Gln, and (iv) Tyr and Phe. The other residues could not be classified. (2) The matrix of distances between amino acids, or scoring matrix, determined from this study, is different from any other published matrix. (3) Except for the distance matrices based on the chemical properties of amino acid side-chains, which can be grouped together, all other published matrices are different from one another. (4) The distance matrix determined in this study seems to be very efficient for aligning distantly related protein sequences.

Amino Acid Sequence↗

PubMed2002

Gas-phase conformations of cationized poly(styrene) oligomers.

The gas-phase conformations of poly(styrene) oligomers cationized by Li+, Na+, Cu+, and Ag+ (M+ PSn) were examined using ion mobility experiments and molecular mechanics/ dynamics calculations. M+PSn ions were formed by MALDI and their ion-He collision cross-sections were measured by ion mobility methods. The experimental collision cross-sections of each M+PS n-mer were similar for all four metal cations and increased linearly with n. Molecular modeling of selected M+ PS oligomers cationized by Li+ and Na+ yielded quasi-linear structures with the metal cation sandwiched between two phenyl groups. The relative energies of the structures were approximately 2-3 kcal/mol more stable when the metal cation was sandwiched near the middle of the oligomer chain than when it was near the ends of the oligomer. The cross-sections of these theoretical structures agree well with the experimental values with deviations typically around 1-2%. The calculations also show that the metal cation tends to align the phenyl groups on the same side of the -CH2-CH- backbone. Calculations on neutral poly(styrene), on the other hand, showed structures in which the phenyl groups were more randomly positioned about the oligomer backbone. The conformations and metal-oligomer binding energies of M+PS are also used to help explain CID product distributions and fragmentation mechanisms of cationized PS oligomers. etry

Cations↗

PubMed2005

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

BACKGROUND: The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. METHODS: The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein. RESULTS: The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. CONCLUSIONS: The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).

Animals↗

PubMed2002

Isolation of Drosophila activin and follistatin cDNAs using novel MACH amplification protocols.

With the genomic sequence of multicellular organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens completed and others to be finished in the near future, the focus has shifted from accumulating sequence information to the prediction and analysis of genes within the completed genomes. Unfortunately, presently available computer programs do not always accurately predict gene structure such as mRNA and translation start sites or intron/exon boundaries. The only way to be certain about a gene's structure is to isolate and characterize its cDNA. Since the screening of libraries is a time-consuming, labor-intensive process that sometimes fails to yield the desired clone, we searched for faster, more efficient ways to isolate cDNAs. In this study, we describe two methods for amplification and isolation of cDNAs from plasmid libraries that requires no hybridization (MACH). With the polymerase chain reaction-based MACH-2 protocol, we present a strategy that requires little DNA sequence information to selectively isolate the longest cDNA variant from plasmid libraries in about 3 days. Our protocols were used to isolate cDNAs for the Drosophila activin and follistatin genes.

Activins↗

PubMed2005

Protein side-chain packing problem: a maximum edge-weight clique algorithmic approach.

"Protein Side-chain Packing" has an ever-increasing application in the field of bio-informatics, dating from the early methods of homology modeling to protein design and to the protein docking. However, this problem is computationally known to be NP-hard. In this regard, we have developed a novel approach to solve this problem using the notion of a maximum edge-weight clique. Our approach is based on efficient reduction of protein side-chain packing problem to a graph and then solving the reduced graph to find the maximum clique by applying an efficient clique finding algorithm developed by our co-authors. Since our approach is based on deterministic algorithms in contrast to the various existing algorithms based on heuristic approaches, our algorithm guarantees of finding an optimal solution. We have tested this approach to predict the side-chain conformations of a set of proteins and have compared the results with other existing methods. We have found that our results are favorably comparable or better than the results produced by the existing methods. As our test set contains a protein of 494 residues, we have obtained considerable improvement in terms of size of the proteins and in terms of the efficiency and the accuracy of prediction.

Algorithms↗

PubMed2002

Comparative modelling of cytochromes P450.

The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.

Amino Acid Sequence↗

PubMed2006

Dendritic saccharide surfactant polymers as antifouling interface materials to reduce platelet adhesion.

Here, we report on the synthesis of dendritic saccharide surfactant polymers as antifouling interface materials to reduce platelet adhesion. An acetal-protected poly(amidoamine) (PAMAM) dendron (5, G = 2) was first synthesized by using aminoacetaldehyde dimethyl acetal (1) as the starting material to provide a monovalent focal structure with dimethyl acetal-protected aldehyde functionality. Maltose dendron (M4, 6) was obtained by reacting the peripheral amine groups of acetal-dendron (5) with maltonolactone. The dendritic surfactant polymers (9) were then synthesized via a two-step method by sequential addition of maltose dendron and hexanal to react with the amine groups on the poly(vinylamine) (PVAm) backbone. Surface activity of the amphiphilic glycopolymers at the air/water interface was demonstrated by reduction in water surface tension. Adsorption of the amphiphilic glycopolymers at the solid/water interface was examined on octadecyltrichlorosilane (OTS)-coated coverslips by water contact angle measurements. A nanoscale understanding of surface-induced self-assembly of the dendritic surfactant polymer on highly oriented pyrolytic graphite (HOPG) was gained using AFM operated in fluid tapping mode. A lateral ordering of adsorbing surfactant polymer was visualized with a pattern in strands 60 degrees out of alignment. The static platelet adhesion tests show that the hexyl side chains can facilitate adsorption of the surfactant polymers onto hydrophobic substrates, while the maltose dendron side chains can provide a dense canopy of protective glycocalyx-like layer as an antifouling interface to reduce platelet adhesion.

Adsorption↗

PubMed2003

Associations between single nucleotide polymorphisms in candidate genes and growth rate in Arctic charr (Salvelinus alpinus L.).

We tested for associations between single nucleotide polymorphisms (SNPs) in five candidate genes allied with the growth hormone axis and the age-specific growth rate of Arctic charr (Salvelinus alpinus L.: Salmonidae). Two large full sib families (N=217 and 95) were created by backcrossing males that were hybrids between two phenotypically divergent populations from Labrador, Canada and from Nauyuk Lake, Canada to females that were from Nauyuk Lake. Measures of individual growth rate (wet weight and fork length) were made three times during a 420-day period after the juveniles were transferred from 4 to 11 degrees C. We then identified SNP markers in 10 proposed candidate genes known to be related to the growth hormone axis. Comparative alignments of amino-acid sequences and nucleotide sequences from other fish species were used to design PCR primers that would amplify 0.5-3 kb DNA regions of the candidate genes. All the individuals in the two backcross families were genotyped for these SNP markers using either polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) or bidirectional amplification of specific alleles (Bi-PASA) approaches. A significant association between a particular SNP allele and early growth was found for the locus containing the growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide genes (GHRH/PACAP2, P=0.00001). We argue that using comparative sequence information to design PCR primers for candidate genes is an efficient method for locating quantitative triat loci in nonmodel organisms.

Animals↗

PubMed1998

Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.

PURPOSE: To isolate and characterize a novel cathepsin gene, as part of the systematic isolation of genes uniquely active in corneal epithelium. METHODS: For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequence tag clones classified as unique to corneal epithelium. Inserted cDNA was introduced into insect cells using a baculovirus expression system, and the secretion of recombinant protein was identified using antisera against a synthetic peptide. Proteolytic activity was determined using bovine serum albumin (BSA) as substrate. The expressions of the novel cathepsin in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The full-length cDNA clone encoded a peptide of 334 amino acids with 82% identity with bovine cathepsin L and 77% identity with human cathepsin L when aligned. The recombinant protein produced in the baculovirus expression system cleaves BSA, and its activity was inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, but not by pepstatin A, phenylmethylsulfonyl fluoride, and EDTA. By RT-PCR, a low level of expression was observed in some other epithelial tissues of ectodermal origin, but only in cornea was it higher than cathepsin L, which is known to be a general lysosomal cathepsin. Cathepsin V protein was detected in human corneal epithelium by western blot analysis, but not in tear fluid. CONCLUSIONS: The amino acid homology and proteolytic activity of the recombinant protein indicate that the novel gene is a new member of the cathepsins that have features of cysteine proteinase. Its uniquely high expression in corneal epithelium strongly implies an important role in corneal physiology.

Amino Acid Sequence↗

PubMed1999

Gene structure and amino acid sequence of Latimeria chalumnae (coelacanth) myelin DM20: phylogenetic relation of the fish.

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2-7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941-1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

Amino Acid Sequence↗

PubMed2001

Exon 2 sequence analysis of a novel HLA-DRB1 allele, DRB1*0314.

We describe the identification of a new DRB1*03 variant in a Caucasian volunteer bone-marrow donor (DR'KW'). The indication for the existence of this new variant arose from contradictory results obtained by serological and several DNA typing methods (SSP, SSOP). Analysis of the exon 2 sequence revealed that the DRB1*0314 contained the characteristic DR3 specific sequence motifs, except for codon 77 (AAC-->ACC) where threonine is substituted for asparagine.

Alleles↗

PubMed2003

Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine.

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Base Sequence↗

PubMed1999

hsp65 sequencing for identification of rapidly growing mycobacteria.

Partial sequencing of the hsp65 gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175-178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium peregrinum, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment. hsp65 sequencing unambiguously differentiated M. chelonae and M. abscessus, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.

Antigens, Bacterial↗

PubMed2003

PROBEmer: A web-based software tool for selecting optimal DNA oligos.

PROBEmer (http://probemer.cs.loyola.edu) is a web-based software tool that enables a researcher to select optimal oligos for PCR applications and multiplex detection platforms including oligonucleotide microarrays and bead-based arrays. Given two groups of nucleic-acid sequences, a target group and a non-target group, the software identifies oligo sequences that occur in members of the target group, but not in the non-target group. To help predict potential cross hybridization, PROBEmer computes all near neighbors in the non-target group and displays their alignments. The software has been used to obtain genus-specific prokaryotic probes based on the 16S rRNA gene, gene-specific probes for expression analyses and PCR primers. In this paper, we describe how to use PROBEmer, the computational methods it employs, and experimental results for oligos identified by this software tool.

Algorithms↗