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Results for “alignment chaining method”

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PubMed1981

The structure of the lentil (Lens culinaris) lectin. Amino acid sequence determination and prediction of the secondary structure.

The subunit structure and complete amino acid sequence of the lectin extracted from Lens culinaris (LcL) seeds was determined. In previous studies, the primary structure of the alpha-chain (Mr = 5,710) was shown to be homologous to the alpha-chain of the lectin from Pisum sativum, the Vicia cracca glucose-specific lectin, and a region in the middle of the concanavalin A sequence (residues 70-121). The complete amino acid sequence of the beta-chain (Mr = 17,572) has been determined from 11 tryptic peptides, 4 peptides derived by chemical cleavage of the beta-chain at its three tryptophan residues, 11 peptides obtained after digestion with Staphylococcus aureus protease, and 5 tryptic peptides from the succinylated polypeptide chain. The extensive homologies by alignment of the alpha- and beta-chains of the L. culinaris lectin with portions of concanavalin A situated between 1 to 45 and 70 to 237, suggest that the L. culinaris and Canavalia ensiformis lectins have evolved from each other. A comparison was made between the secondary structure of the C. ensiformis lectin and the probable secondary structure of the L. culinaris lectin as predicted by two different methods. The results indicate that the folding of these two polypeptides has been particularly well conserved during evolution. It is suggested that the L. culinaris lectin is synthesized as a single polypeptide chain and cleaved subsequently into two or possibly three fragments, two of which would be alpha and beta and the third a fragment homologous to portion 46 to 69 in concanavalin A. As in favin, the amino acids postulated to be involved in the formation of the hydrophobic cavity and the sugar and metal binding sites are highly conserved in L. culinaris lectin.

Amino Acid Sequence↗

PubMed1995

HLA-DQA1 typing in Danes by two polymerase chain reaction (PCR) based methods.

A total of 280 persons were HLA-DQA1 typed by two different polymerase chain reaction (PCR) based methods; (i) a reverse dot-blot (RDB) method, which can differentiate between six alleles, and (ii) a combined PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific amplification (ASA) method, which together recognise eight alleles. In 146 unrelated Danish individuals, the HLA-DQA1 alleles were in Hardy-Weinberg equilibrium. For identity testing, the power of discrimination (PD) of HLA-DQA1 was 0.932 with the RDB method and 0.942 with the PCR-RFLP/ASA method. For paternity testing, the theoretical chance of exclusion in HLA-DQA1 of non-fathers was 0.634 with the RDB method and 0.660 with the PCR-RFLP/ASA method.

Alleles↗

PubMed2002

Cloning of interleukin-4 delta2 splice variant (IL-4delta2) in chimpanzee and cynomolgus macaque: phylogenetic analysis of delta2 splice variant appearance, and implications for the study of IL-4-driven immune processes.

The human interleukin-4 ( IL-4) gene produces an exon 2-lacking alternative splice variant, termed IL-4delta2, and described as a naturally occurring antagonist of IL-4-driven activity. We report the isolation of an IL-4delta2 cDNA from chimpanzee ( Pan troglodytes) bone marrow samples and cynomolgus macaque ( Macaca fascicularis) activated peripheral lymph node cells. The complete IL-4 cDNA sequence from chimpanzee is also provided for the first time. The phylogenetic analysis of several known IL-4 sequences revealed a highly conserved structure of coding regions among primates, suggesting that alternative IL-4 transcript splicing may be a process shared by other simian and potentially pro-simian species as well. Extension of the study to other mammalian species led us to the assumption that generation of IL-4 splice variants may be common to primates, lagomorphs (rabbit), and rodents of the sciuridae family (woodchuck), but is unlikely to occur in mice and rats (muridae), for which IL-4 splice variants have indeed never been described. Potential implications of alternatively spliced cytokine products with possible antagonistic or competitive inhibitory function, for the choice of suitable animal models of IL-4-regulated immune processes, are discussed. This study also indicates the importance of considering alternative splicing when defining cytokine bioassays, most particularly in the present context of transcriptomics, involving the generalization of sequence-based detection methods such as quantitative reverse transcription PCR.

Alternative Splicing↗

PubMed2003

Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field.

Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.

Ascomycota↗

PubMed2004

Performance of an iterated T-HMM for homology detection.

MOTIVATION: Much information about new protein sequences is derived from identifying homologous proteins. Such tasks are difficult when the evolutionary relationships are distant. Some modern methods achieve better results by building a model of a set of related sequences, and then identifying new proteins that fit the model. A further advance was the development of iterative methods that refine the model as more homologs are discovered. These methods are generally limited by ad hoc methods of sequence weighting, neglect of underlying evolutionary relationships and the representation of the set with a single one-size-fits-all model. These limitations are avoided through the use of a Tree hidden Markov model (T-HMM) approach. Our previous work described how a non-iterative version of the T-HMM method could identify distant homologs with superior performance compared with other non-iterated approaches, and described how this method was particularly appropriate for being implemented as an iterative algorithm. RESULTS: We describe an iterative version of the T-HMM algorithm, and evaluate its performance for the detection of distant homologs. Significant improvement over other commonly used methods is found. AVAILABILITY: The software (C++, Perl) is available from the corresponding author.

Algorithms↗

PubMed2006

Early assessment of the likely cost-effectiveness of a new technology: A Markov model with probabilistic sensitivity analysis of computer-assisted total knee replacement.

OBJECTIVES: The objective of this study is to apply a Markov model to compare cost-effectiveness of total knee replacement (TKR) using computer-assisted surgery (CAS) with that of TKR using a conventional manual method in the absence of formal clinical trial evidence. METHODS: A structured search was carried out to identify evidence relating to the clinical outcome, cost, and effectiveness of TKR. Nine Markov states were identified based on the progress of the disease after TKR. Effectiveness was expressed by quality-adjusted life years (QALYs). The simulation was carried out initially for 120 cycles of a month each, starting with 1,000 TKRs. A discount rate of 3.5 percent was used for both cost and effectiveness in the incremental cost-effectiveness analysis. Then, a probabilistic sensitivity analysis was carried out using a Monte Carlo approach with 10,000 iterations. RESULTS: Computer-assisted TKR was a long-term cost-effective technology, but the QALYs gained were small. After the first 2 years, the incremental cost per QALY of computer-assisted TKR was dominant because of cheaper and more QALYs. The incremental cost-effectiveness ratio (ICER) was sensitive to the "effect of CAS," to the CAS extra cost, and to the utility of the state "Normal health after primary TKR," but it was not sensitive to utilities of other Markov states. Both probabilistic and deterministic analyses produced similar cumulative serious or minor complication rates and complex or simple revision rates. They also produced similar ICERs. CONCLUSIONS: Compared with conventional TKR, computer-assisted TKR is a cost-saving technology in the long-term and may offer small additional QALYs. The "effect of CAS" is to reduce revision rates and complications through more accurate and precise alignment, and although the conclusions from the model, even when allowing for a full probabilistic analysis of uncertainty, are clear, the "effect of CAS" on the rate of revisions awaits long-term clinical evidence.

Arthroplasty, Replacement, Knee↗

PubMed1996

A comparative analysis of prostate-specific antigen gene sequence in benign and malignant prostate tissue.

OBJECTIVES: Different molecular forms of prostate-specific antigen (PSA) appear to be expressed by benign prostatic hyperplasia (BPH) compared with prostate cancer. These differences are not well understood and may arise from aberrant RNA splicing, altered protein glycosylation, or variant PSA complexing to macroglobulins. To our knowledge, a direct comparison of PSA mRNA sequences in BPH versus prostate cancer to account for these differences has not been reported. The purpose of this study was to compare the complete PSA mRNA gene sequences in benign and malignant prostate tissue to determine whether altered PSA phenotypes are a result of gene mutations and to compare the published PSA sequences. METHODS: Total RNA was extracted from 17 prostate specimens from 8 patients, including matched benign and malignant prostate tissue in 6 patients. The samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) of the PSA coding sequence and part of the 3' untranslated region. Directed DNA sequencing was performed on these fragments. RESULTS: The benign and malignant prostate tissue cDNA sequence data of both strands were aligned and a computer analysis revealed 100% match with no evidence of mutation in prostate cancer compared to normal tissue. Sequence analysis did not reveal point mutations or aberrant splicing in any of the samples, including the matched malignant and nonmalignant tissues. Comparison with published sequences revealed infrequent and inconsistent sequence differences. CONCLUSIONS: These findings suggest that the PSA gene expressed in malignant prostate tissue is the wild type. PSA structural alterations previously reported in the literature may occur through post-transitional mechanisms. A detailed understanding of the possible differences in the PSA gene sequence is essential as we develop newer techniques that utilize RT-PCR to perform molecular diagnosis and staging of prostate cancer.

Base Sequence↗

PubMed2026

Evaluation of one-step amplicon-based targeted enrichment for SARS-CoV-2 whole-genome sequencing using the Midnight amplicon scheme.

Genomic surveillance proved invaluable during the COVID-19 pandemic for tracking SARS-CoV-2 variants and guiding outbreak responses, underscoring the ongoing need to reduce whole-genome sequencing (WGS) costs and improve workflow efficiency to ensure accessibility in resource limited settings. Here, we evaluated a one-step reverse transcription polymerase chain reaction (RT-PCR) approach using the Midnight V2 primer scheme for targeted amplification of the SARS-CoV-2 genome, assessed its compatibility with Illumina sequencing, and compared its performance to a well-established two-step method. Initially, we determined optimal RT-PCR reaction conditions using the Midnight V2 primer panel for the one-step RT-PCR kit and scaled reaction volumes for both RT-PCR and library preparation. Clinical specimens (n = 53) that had undergone routine WGS for surveillance purposes using the established two-step RT-PCR method were compared using the one-step RT-PCR assay. For samples with genome completeness greater than 70%, both methods gave comparable results with similar sequence coverage and 100% concordance for lineage assignment. Further investigation revealed a higher percentage of reads aligning to the SARS-CoV-2 genome with a greater depth of coverage using the one-step method compared to the two-step method. Finally, analysis of scaled one-step and library reaction volumes revealed significant cost savings for samples undergoing WGS. Overall, the results presented here verify the accuracy and reproducibility of one-step targeted amplification and offer an efficient and cost-effective workflow for routine SARS-CoV-2 genomic surveillance.

Humans↗

PubMed2004

Direct detection of bacterial pathogens in brain abscesses by polymerase chain reaction amplification and sequencing of partial 16S ribosomal deoxyribonucleic acid fragments.

OBJECTIVE: To evaluate the feasibility of detecting bacterial pathogens directly from the clinical brain abscess specimens by polymerase chain reaction (PCR) amplification and sequencing of bacterial 16S ribosomal deoxyribonucleic acid (rDNA). METHODS: A total of 14 specimens were tested by both culture and PCR amplification, targeting the full-length or a partial region of 16S rDNA. 16S rDNA is known to be conserved in bacteria. Sequencing of partial-length and full-length 16S rDNA was performed. The sequence data were compared with known sequences of 16S rDNA in the National Center for Biotechnology Information GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithm. The species with the best match of similarity were regarded as the pathogenic species in the samples. We also developed a Streptococcus-specific multiplex PCR analysis for identifying members of the Streptococcus species, the most common pathogen of brain abscesses. RESULTS: The 10 culture-positive specimens were all PCR-positive for partial 16S rDNA, but only seven were positive for full-length 16S rDNA amplification. Bacterial DNA was not detected in the remaining four specimens with a negative culture. Species identification by phenotypes from culture was in agreement with that by sequencing results of partial-length (or full-length) 16S rDNA. The Streptococcus-specific PCR analysis could detect Streptococcus species correctly in one step. CONCLUSION: Bacterial 16S rDNA sequences provide reliable clues to the identification of unknown pathogens. PCR analysis of 16S rDNA and sequencing may identify pathogens to the species level directly from brain abscesses. This approach is rapid and is useful especially in the identification of slow-growing and fastidious organisms.

Adolescent↗

PubMed2005

PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae).

A polymerase chain reaction with the restriction fragment length polymorphism (PCR-RFLP) method using universal primers complementary to the conserved region of the cytochrome b gene (cyt b) of the mitochondrion DNA (mtDNA) of vertebrates was applied to the identification of the origin of blood meals in tsetse flies. Blood samples from ten potential tsetse hosts of the family bovidae (cattle, water buffalo, red buffalo, waterbuck, springbok, goat, sheep, sable antelope, oryx and dik-dik) were included in this study. Sites for appropriate restriction endonucleases cuts were chosen by pairwise alignment of the amplified 359 bp fragments. A flow chart of endonucleases digestion using three restriction enzymes (e.g. TaqI, AluI and HindII) for the unequivocal identification of the respective bovid species was developed. A number of additional non-specific DNA fragments attributed to the co-amplification of cytochrome b pseudogenes were observed in some species (e.g. in red buffalo and dik-dik after digestion with AluI) but did not hamper assignment of bovid species. The detection rate of host DNA in tsetse by PCR-RFLP was 100, 80, 60 and 40% at 24, 48, 72 and 96 h after in vitro feeding, respectively. Identification of the last blood meal was possible even when tsetse had previously fed on different hosts.

Animals↗

PubMed1994

Prediction of protein side-chain conformations from local three-dimensional homology relationships.

A method for predicting the conformations of protein side-chains, starting from main-chain co-ordinates alone, is described. The method involves the comparison of the local environment of each residue whose side-chain conformation is to be predicted with a database of local environments for the same residue type constructed from an analysis of high-resolution protein structures. Local environments are described in terms of the residue type and location in space of residues that interact with the side-chain of interest. The best (most three-dimensionally homologous) few matches to each residue are then input to a Monte-Carlo procedure to give a final predicted structure. The method has been tested on a selection of eight proteins, ranging in size from 46 to 323 amino acid residues. The average side-chain atom root-mean-square deviation between the actual and predicted structures is 1.71 A taken over all residues, and 1.00 A if restricted to buried residues. Over all residues, an average of 59.8% of all side-chain dihedral angles are predicted within +/- 30 degrees of the crystal structure values. Considering buried residues only, this rises to 79.6%.

Aprotinin↗

PubMed1998

An Off-resonance Rotating Frame Relaxation Experiment for the Investigation of Macromolecular Dynamics Using Adiabatic Rotations

15N off-resonance rotating frame relaxation can be applied to the study of internal dynamics in proteins in the millisecond to microsecond regime. We show that the performance of existing methods can be improved by application of simultaneous amplitude and phase-modulated adiabatic RF pulses to align the nuclear spin magnetization with the off-resonance spin-lock field for all the spins under investigation. Application of this technique to the 269-residue serine protease PB92 allowed the measurement of 15N off-resonance rotating frame relaxation rates for all nonoverlapping residues in the protein, including the arginine side chains, encompassing a chemical shift range of 50 ppm. Simulations indicate that by use of the proposed adiabatic RF pulses rotating frame relaxation rates can be obtained for magnetization vectors aligned at arbitrary angles with the static field. Copyright 1998 Academic Press.

Journal Article↗

PubMed1997

Characterisation of gamma herpesviruses in the horse by PCR.

A polymerase chain reaction (PCR) based on a combination of oligonucleotide primers selected using the octamer frequency disparity method with primers specific for EHV-5 (described by other authors) recognized all of a series of gamma herpesvirus field isolates. This PCR produced only three fragments: (1) one EHV-2-specific; (2) one EHV-5-specific; and (3) a fragment that occurred alone or in combination with the other two. Cloning and sequencing of four different isolates yielding only the last PCR product showed that this corresponds to a deletion/insertion mutant of EHV-2. The fact that this mutant was also plaque-purified from a culture producing all three PCR fragments demonstrated that the virus producing this fragment was distinct from the other two and that this specific DNA fragment was not an artefact due to PCR amplification. These data show that equine gamma herpesviruses are genetically more heterogeneous than previously assumed. The PCR failed to directly detect gamma herpesviruses from the DNA extracted from the same starting material used for the isolation of gamma herpesvirus by cocultivation with indicator cells. This demonstrates that the most reliable method for detection of equine gamma herpesviruses is the cocultivation with indicator cells.

Animals↗

PubMed1998

Phylogenetic position of symbiotic protist Dinenympha [correction of Dinemympha] exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha.

The phylogenetic position of the symbiotic oxymonad Dinenympha exilis, found in the hindgut of the lower termite Reticulitermes speratus, was determined by analysis of translation elongation factor 1 alpha (EF-1 alpha). cDNA corresponding to a major part of the amino acid coding region of EF-1 alpha mRNA was amplified by the reverse transcription polymerase chain reaction (RT-PCR) method from total mRNA of termite hindgut microorganisms without cultivation. The product was cloned into a plasmid vector, pGEM-T, and the clones were isolated and sequenced. One of the EF-1 alpha clones isolated was assigned to the protist D. exilis by whole-cell in-situ hybridization using a specific oligonucleotide probe with enzymatic signal amplification. The deduced amino acid sequence was aligned with those of other eukaryotic and archaeabacterial EF-1 alpha s, and the phylogenetic relationships among early branching eukaryotes were inferred by using the distance matrix method and the maximum parsimony method. The phylogenetic analysis indicated that the D. exilis offshoot occurred before mitochondria-containing organisms and D. exilis branched out after the diplomonads clade. These results indicate that the oxymonad D. exilis is one of the early branching organisms and suggest that the oxymonads form a lineage independent of other early branching organisms.

Amino Acid Sequence↗

PubMed2000

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex.

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.

Base Sequence↗

PubMed2004

Study on the polymorphism of bovine lactoferrin gene and its relationship with mastitis.

Mastitis is a major cause of economic loss to the dairy industry. Lactoferrin (Lf) is known to contribute to resistance against bacterial infections. Hence, we decided to characterize the relevance between mastitis resistance and the variants of Lf gene. By using PCR-SSCP, five fragments within 5' region and all exons of bovine lactoferrin gene were amplified and identified the nucleotide diversity. For the five segments within the 5'-region: Lf5'-1, Lf5'-2, Lf5'-3, Lf5'-4, and Lf5'-5 from upstream to downstream, we found that three had base variation. Totally, mutations were observed in Lf5'-1, Lf5'-3, and Lf5'-5, exons 4, 8, 9, 11, 15, and intron 4. We analyzed the effects of all mutated loci on milk production traits with least squares method.

Animals↗

PubMed2006

Cloning and identification of porcine SMPX differentially expressed in F1 crossbreds and their parents.

In order to investigate porcine heterosis on the molecular basis, Large White (L), a European purebred, and Meishan (M), a Chinese indigenous purebred, were hybridized directly and reciprocally to produce F1 hybrids, Large WhitexMeishan (LM) and MeishanxLarge White (ML) pigs. Using mRNA differential display, we found an expression sequence tag (EST) differentially expressed in F1 hybrids and their parents, designated as EST55, which was homologous to human and murine skeletal muscle protein (SMPX), and the full-length cDNA of porcine SMPX was cloned by the rapid amplification of cDNA end (RACE) method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 86 amino acid residues encoding a nuclear location signal peptide, two overlapping casein kinase II phosphorylation sites and one N-glycosylation site with theoretical molecular weight of 9.3 kDa. Alignment analysis revealed that the deduced protein sequence shared 94%, 83% and 78% homology with that of its human, mouse and rat counterparts, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that it was expressed predominantly in skeletal and heart muscles, whereas at a moderate level in backfat, spleen, stomach and uterus tissues. Two single nucleotide polymorphism (SNPs), located in 5'- and 3'-untranslated region (UTR), respectively,were identified by PCR and sequencing. Phylogenetic tree and the secondary structure prediction were also performed. The possible relationship between porcine SMPX and heterosis was discussed.

Amino Acid Sequence↗

PubMed2001

[Cloning of the mouse Doc-1R gene by genomic walking].

OBJECTIVE: To obtain the genomic sequences of the mouse Doc-1R gene. METHODS: Gene-specific primers were designed and synthesized based on the cDNA sequences of the mouse Doc-1R gene. With the use of genomic walking strategy, the mouse genomic walking library was amplified by the polymerase chain reaction(PCR). Mouse genomic library constructed with a special adaptor was utilized as a template to amplify the desired fragment by nested PCR. RESULTS: A desired fragment of 1.5 kb was obtained. Sequence analysis of the desired fragment confirmed that the genomic cloning of the Doc-1R gene was successful. This gene contains four exons and three introns. All of the splice donor/acceptor site sequences are in accordance with the consensus 'GT-AG' rule. CONCLUSION: The genomic walking strategy is simple, efficient and reliable; it is an ideal method of cloning genomic fragments.

Animals↗