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Results for “alignment chaining method”

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PubMed1992

Evolutionary divergence plots of homologous proteins.

A simple and efficient method is described for analyzing quantitatively multiple protein sequence alignments and finding the most conserved blocks as well as the maxima of divergence within the set of aligned sequences. It consists of calculating the mean distance and the root-mean-square distance in each column of the multiple alignment, averaging the values in a window of defined length and plotting the results as a function of the position of the window. Due attention is paid to the presence of gaps in the columns. Several examples are provided, using the sequences of several cytochromes c, serine proteases, lysozymes and globins. Two distance matrices are compared, namely the matrix derived by Gribskov and Burgess from the Dayhoff matrix, and the Risler Structural Superposition Matrix. In each case, the divergence plots effectively point to the specific residues which are known to be essential for the catalytic activity of the proteins. In addition, the regions of maximum divergence are clearly delineated. Interestingly, they are generally observed in positions immediately flanking the most conserved blocks. The method should therefore be useful for delineating the peptide segments which will be good candidates for site-directed mutagenesis and for visualizing the evolutionary constraints along homologous polypeptide chains.

Amino Acid Sequence↗

PubMed1992

Multiplex random priming of internal restriction fragments for DNA sequencing.

An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.

Automation↗

PubMed2025

Molecular Identification and Genotyping of Blastocystis Spp. In Children with Clinical Symptoms in Southeast Iran Using PCR-Sequencing Method.

Blastocystis spp. is a zoonotic anaerobic parasite that has been identified in the large intestine of humans and many vertebrates. It is predominantly encountered in individuals with frequent contact with animals. The present study aims to identify the prevalence of Blastocystis spp. and its common genotypes in children with clinical symptoms of diarrhea in the city of Zahedan, located in the southeast of Iran. A cross-sectional descriptive study was conducted on 60 children under ten years of age with gastrointestinal symptoms, especially diarrhea. Following the collection of samples, stool samples were subjected to direct stool testing for the initial diagnosis. Following this, a microscopic diagnosis was made, after which DNA was extracted and a Polymerase Chain Reaction (PCR) test with a small subunit ribosomal RNA (SSU rRNA) gene target was performed. The PCR products were then purified and sequenced. The resulting nucleotide sequences were then subjected to a thorough review using Chromas biotechnology software version 2.4 and CLC genomic work bench software 11. The alignment of the nucleotide sequences was subsequently facilitated by utilizing the BLAST database, and these sequences were then compared with the reference genotypes of Blastocystis spp. that are stored within the gene bank. The genotyping of the sequences was conducted using CLC genomic work bench software 11, and a phylogenetic tree was constructed using MEGA7 software with the Neighbor-Joining statistical method, which applied the Kimura 2-parameter method. Out of the 60 cases that were examined, 5 children (8.33%) were found to be positive by direct microscopic and PCR tests, where a 500 (479) bp fragment in the SSU-rRNA target was detected. Subsequent genetic analysis identified four distinct subtypes, including subtypes 1, 2, 3, and 5. The percentage of nucleotide identity with the sequences in the gene bank was found to be between 93 and 100%. Given the presence of subtypes 3 and 5 in the study and the evidence of their zoonotic nature, it can be concluded that examining parasite dynamics and epidemiological principles can be effective in the control strategy.

Blastocystis↗

PubMed1991

[Homology of Chinese, Japanese, and American strains of hepatitis C virus].

As we know, the main HCV strain in Japan (HCV-J 4) is highly diverse in sequence from the original American strain (HCV-US). By comparing the sequences of these two strains, a region, located in the 3'-terminus of the putative E gene in HCV genome, was selected for PCR amplification. In this region, the Japanese strain showed high diversity from the American strain. From 4 patients, 8 isolates with the length of 277 bp, were cloned and sequenced. The homology of these 8 clones was high, indicating that these clones were in the same strain. This strain might be the major strain in China, named HCV-CHN1 in this paper. Since there were still many varieties in these 8 clones, HCV CHN1 could be possibly divided into 3 substrains. HCV-CHN1 was also found to be similar to HCV-J 4, with nucleotide homology of 83.4-86.3%, and amino acid homology of 78.3-88.0%, but remote from HCV-US, with homologies of only 68.6-72.6% and 69.6-73.9%, respectively. These results might be of paramount significance in developing the detection method for HCV infection and in preparing HCV vaccine.

Amino Acid Sequence↗

PubMed2026

The knee-ankle link: impact of knee varus severity on distal joint malalignment and concomitant pathologies.

BACKGROUND: Knee varus deformity is traditionally managed as an isolated joint pathology; however, persistent distal symptoms following proximal realignment suggest a more extensive kinetic chain dysfunction. The degree to which knee varus severity dictates distal malalignment and secondary pathologies remains poorly quantified in the current literature. METHODS: This systematic review and meta-analysis were conducted in accordance with PRISMA 2020 guidelines (PROSPERO: CRD420261363327). A comprehensive search of PubMed, Embase, Web of Science, and the Cochrane Library was performed from inception to April 2026. Studies examining the relationship between knee varus (HKA angle) and radiographic distal alignment or pathologies were included. Data synthesis utilized random-effects models, with prevalence analyzed via generalized linear mixed models (GLMM). RESULTS: Fourteen studies were included in the final synthesis. While pooling of continuous radiographic parameters was limited by high statistical heterogeneity in Talar Tilt (I2 = 96.5%), individual large-cohort data (Huang et al.) indicated that severe knee varus (HKA > 10°) was associated with increased odds of concomitant ankle osteoarthritis (OR 2.29; 95% CI 1.28-4.11) and a specific cohort prevalence of 37.1%. Furthermore, single-arm prevalence data revealed divergent trends across different study populations, with compensatory hindfoot valgus reaching 69.9% in some cohorts and rigid varus up to 63.9% in others. CONCLUSIONS: Severe genu varum is associated with distal kinetic chain alterations and concomitant ankle pathologies. However, due to the extreme heterogeneity and divergent distal adaptations observed across different cohorts, standardized knee-centric protocols may be insufficient. Further longitudinal and interventional studies are required to establish phenotype-specific rehabilitation guidelines.

Humans↗

PubMed1976

Isolation and amino acid sequence of a neurotoxic phospholipase A from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.

Amino Acid Sequence↗

PubMed1990

The vaccinia virus HindIII K fragment encodes a novel protein belonging to the serpin superfamily.

The 1983-base pair nucleotide sequence of the EcoRI-HindIII fragment of vaccinia virus Tiantan strain HindIII K clone is determined by the dideoxy chain termination method. A search in the NBRF protein sequence database using FASTA and other microcomputer programs reveals that several proteins belonging to the serpin (serine protease inhibitor) superfamily have striking similarities to the protein encoded by the HindIII K1 ORF. On the basis of the dot-matrix analysis and sequence alignment, the K1-encoded protein is shown as a novel member of the serpin superfamily. The putative reactive site and switch sequence of this novel serpin are then compared with those of other serpins. The probable evolutionary and possible functional relationships are discussed.

Base Sequence↗

PubMed1991

Detection of common three-dimensional substructures in proteins.

We present a fully automatic algorithm for three-dimensional alignment of protein structures and for the detection of common substructures and structural repeats. Given two proteins, the algorithm first identifies all pairs of structurally similar fragments and subsequently clusters into larger units pairs of fragments that are compatible in three dimensions. The detection of similar substructures is independent of insertion/deletion penalties and can be chosen to be independent of the topology of loop connections and to allow for reversal of chain direction. Using distance geometry filters and other approximations, the algorithm, implemented in the WHAT IF program, is so fast that structural comparison of a single protein with the entire database of known protein structures can be performed routinely on a workstation. The method reproduces known non-trivial superpositions such as plastocyanin on azurin. In addition, we report surprising structural similarity between ubiquitin and a (2Fe-2S) ferredoxin.

Algorithms↗

PubMed1990

A new concept of fibrin formation based upon the linear growth of interlacing and branching polymers and molecular alignment into interlocked single-stranded segments.

In a previous electron microscopic study of early fibrin polymers processed by freeze drying and rotatory shadowing, a large proportion of loosely constructed, frequently branching linear molecular chains was observed; their structural organization was inconsistent with a half-staggered double-stranded model for fibrin polymerization. These conflicting results prompted us to investigate the structure of early fibrin polymers prepared according to a large variety of methods currently used for electron microscopy of macromolecules. By use of a systematic random sampling procedure, fibrin polymers were photographically recorded. They were classified according to their morphological form, and the frequency of occurrence of each configuration was determined. Half-staggered double-stranded forms accounted for less than 1% of all types encountered. Interpretation of the structural organization manifested in the diverse polymer forms observed necessitated the construction of a new interlocked single-strand model for fibrin polymerization. The fibrin polymerization process combines simultaneous propagation of linear growth, branching, and lateral interlocking (leading to lateral association), resulting in the rapid formation of a fibrin network. The structural pattern developing during growth of fibrin polymers appears to be determined principally by the enzymatic mechanism and not solely by the intrinsic molecular structure of fibrinogen. The validity of the interlocked single-strand model was tested by selective fibrinopeptide-B-releasing experiments. Under such activation conditions, the polymer forms predicted according to this and the half-staggered double-strand models should differ; the structures observed were indeed consistent with the interlocked single-strand hypothesis. The compatibility of existing data with this model is discussed.

Fibrin↗

PubMed1988

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.

Amino Acid Sequence↗

PubMed1979

[Hemoglobins, XXV. Hemoglobin (erythrocruorin) CTT III from Chironomus thummi thummi (Diptera). Primary structure and relationship to other heme proteins (author's transl)].

The amino acid sequence analysis of hemoglobin (erythrocruorin) CTT III from Chironomus thummi th. (Diptera) has been checked with automatic methods and completed. The protein chain consists of 136 amino acids and contains a neutral exchange isoleucine/threonine in position 57. The molecular weight of the heme protein (Thr) is 15400. The primary structure gives the chemical basis for the refinement of the X-ray structure and the understanding of the mechanism of the Bohr effect in this monomeric hemoglobin. A homologous alignment to vertebrate globins is reported. The resulting data for the phylogeny of proto-and deuterostomian animals and the function of this hemoglobin are discussed.

Amino Acid Sequence↗

PubMed1992

Isolation and sequence analysis of the small subunit ribosomal RNA gene from the euryhaline yeast Debaryomyces hansenii.

The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced. After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D. hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis. It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.

Base Sequence↗

PubMed1974

The primary structure of high density apolipoprotein-glutamine-I.

The major protein constituent of human plasma high density lipoproteins has been isolated and its complete amino-acid sequence determined. The protein, designated apolipoprotein-glutamine-I by the presence of carboxyl-terminal glutamine, is a single polypeptide chain of 245 amino-acid residues, including three residues of methionine. The protein is devoid of cysteine, cystine, and isoleucine. Cleavage of apolipoprotein-glutamine-I with cyanogen bromide yields four fragments with 94, 90, 36, and 25 amino acids. The amino-acid sequence of each fragment was determined by conventional methods, with proteolytic digestion with trypsin, chymotrypsin, and thermolysin. The alignment of the cyanogen bromide fragments was determined by the isolation of the methionine-containing tryptic peptides from apolipoprotein-glutamine-I. Inspection of the sequence of apolipoprotein-glutamine-I suggests an interesting distribution of amino acids that may account for its helical structure and its ability to bind and transport lipid.

Amino Acid Sequence↗

PubMed1977

Structural studies on the coat protein of alfalfa mosaic virus. The complete primary structure.

The complete amino acid sequence of the coat protein of alfalfa mosaic virus (strain 425) is reported. Sequence determinations were mainly performed on peptides obtained from fragmentation by cyanogen bromide and trypsin. Both manual and automatic sequence methods were used. Some refinements of the solid-phase Edman degradation were introduced. The final alignment of the peptides was established by means of alternative cleavage methods, such as limited tryptic digestion of intact virus particles, tryptic digestion after blockage of lysine residues and chymotryptic digestion. The coat protein consists of 220 amino acid residues corresponding to a molecular weight of 24252. A remarkable clustering of basic residues occurs in the N-terminal part of the protein chain. Several internal hydrophobic clusters and a strongly acidic site at the C-terminus can be observed. Two regions of sequence homology (12 residues) were found. Some features of the secondary structure are predicted.

Amides↗

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↗

PubMed1992

Partial DNA cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease.

The cloning and sequencing of an Eco RI-PstI fragment derived from the replicative form of a canine parvovirus (CPV) vaccine strain are reported. The variability of the 5' end of NS 1 protein gene in the genome is confirmed by comparison with previously determined DNA sequences. A 15 nucleotide deletion was also observed in this vaccine strain. In order to improve CPV diagnosis, radioactively labelled RNA or DNA and biotin labelled DNA obtained by random priming of the recombinant plasmid were used as probes mainly on gut or stool samples from naturally infected dogs. Results of filter hybridization correlated well with histopathological diagnosis of parvovirus infection and with hemagglutination tests performed on dog faeces. We propose that nucleic acid hybridization may be an alternative diagnostic method to ascertain the presence of CPV, especially in frozen samples.

Amino Acid Sequence↗

PubMed1991

Playing with blocks: some pitfalls of forcing multiple alignments.

Block alignments of multiple amino acid sequences are useful representations of regions thought to share common ancestry and function. Often the block alignments are motivated by the expectation that a protein of interest is similar in function to members of a family of proteins. However, when alignments are forced by using ad hoc methods, it is often difficult to decide whether the proposed relationship is valid. Visual examination can be deceptive, especially when alignments are not carried out in the context of controls subjected to similar procedures. Even computer-aided methods can be misleading when biases are introduced. To illustrate some of the problems that can arise, a few examples from the literature are analyzed. It is concluded that when standard methods fail to find an interesting block alignment unaided by human intervention, then the result should be regarded with caution.

Amino Acid Sequence↗

PubMed1992

Primary structure of a Thomsen-Friedenreich-antigen-specific lectin, jacalin [Artocarpus integrifolia (jack fruit) agglutinin]. Evidence for the presence of an internal repeat.

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

Amino Acid Sequence↗