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PubMed1994

Phylogenetic relationships of 10 grass species: an assessment of phylogenetic utility of the internal transcribed spacer region in nuclear ribosomal DNA in monocots.

Entire sequences of the internal transcribed spacers (ITSs) and 5.8S subunit of nuclear ribosomal DNA (nrDNA) were obtained from nine grass species by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified DNA fragments. These sequences from subfamily Pooideae (Triticum aestivum, Crithodium monococcum, Sitopsis speltoides, Hordeum vulgare, Secale montanum, Avena longiglumis, Bromus inermis, Brachypodium distachyon) and subfamily Panicoideae (Sorghum bicolor) together with published ITS sequence of rice (Oryza sativa, Bambusoideae) were analyzed using Wagner parsimony (PAUP) and the neighbor-joining distance method to assess the phylogenetic utility of ITS sequences at various taxonomic levels. Among the aligned sequences that ranged from 588 to 603 nucleotides in length, 118 of 269 variable sites contained potential phylogenetic information. A member of Bromus, B. inermis, was the sister taxon to the Triticeae species. Brachypodium was more distantly related to Triticeae than was Bromus or Avena. These data, with Oryza sativa as the outgroup, indicate monophyly of the Pooideae species and of the members of the tribe Triticeae within Pooideae. Phylogenetic trees of the 10 grass species generated from the ITS sequence data were in general agreement with phylogenies based on molecular data from ribosomal RNA (rRNA) and chloroplast DNA (cpDNA) of similar grass taxa. This study reaffirms that sequences of the ITS region are useful for phylogenetic inference among closely related monocot species.

Base Sequence↗

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↗

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↗

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↗

PubMed1998

Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.

Base Sequence↗

PubMed1998

A major component approach to presenting consensus sequences.

MOTIVATION: Summarizing and displaying the information contained in a set of aligned sequences is an important aid to identifying patterns within the sequences. A variety of forms of consensus sequences have been used previously to provide this information. However, these methods can cause a loss of information or introduce ambiguities into the consensus sequence, and some graphical approaches may become difficult to interpret due to visual distortion. RESULTS: We have developed a method to present a more precise and graphically clear view of a consensus sequence by using an approach based on defining the major components at each position in a sequence set. The major components are given in an ordered list and their frequencies are shown as histograms which can be colour coded to reflect conservative groupings. Minor components, a one-line character-based consensus sequence and information statistics can also be presented. As well as identifying the dominant sources of variation and conservation in the sequence set, the method also enables similarities and differences between subgroups of a sequence set to be readily assessed. AVAILIABILITY: On request from the authors. CONTACT: bcsmith@usthk.ust.hk, hxue@usthk. ust.hk

Amino Acid Sequence↗

PubMed1997

Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8.

A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.

Base Sequence↗

PubMed1998

Application of a novel and fast information-theoretic method to the discovery of higher-order correlations in protein databases.

We present a fast, discrete data-mining approach to the problem of finding kappa-tuples of correlated amino acid residues in protein sequence data. When sets of sequence-distant sites display high mutual information, they may bespeak important structural or functional features. Our novel methodology overcomes the limitations of previous methods which examined only single-residue features or pairwise interactions.

AIDS Vaccines↗

PubMed1994

Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms.

The identification and taxonomy of papillomaviruses has become increasingly complex, as approximately 70 human papillomavirus (HPV) types have been described and novel HPV genomes continue to be identified. Methods and corresponding DNA sequence data bases were designed for the reliable identification of mucosal HPV genomes from clinical specimens. HPVs are identified by the amplification of a fragment of the L1 region by consensus primer polymerase chain reaction (PCR) and subsequent hybridization or restriction fragment length polymorphism analysis. L1 PCR fragments may be further characterized by nucleotide sequencing. Conservation of 30 (of 151) predicted amino acids identifies HPV genomic fragments, and nucleotide sequence alignments allow calculation of their phylogenetic relatedness. Sequence differences > 10% from any known HPV type suggest a novel HPV type. Phylogenetic relationships with known HPV types may permit predictions of biology. With these criteria, 10 PCR fragments were identified that would qualify as new genital HPV types after complete genomic isolation.

Amino Acid Sequence↗

PubMed1993

The complete amino acid sequence of momordin-a, a ribosome-inactivating protein from the seeds of bitter gourd (Momordica charantia).

The complete amino acid sequence of momordin-a, a ribosome-inactivating protein from the seeds of bitter gourd, has been analyzed. Twenty-two peptides were isolated from the tryptic digest of momordin-a and sequenced by the DABITC/PITC double coupling method. The alignment of these tryptic peptides was done by analyzing the amino acid sequences of the peptides derived from chymotryptic digestion and cyanogen bromide cleavage of momordin-a as well as V8 protease-digestion of the CNBr fragment. Momordin-a consisted of 250 amino acid residues and carbohydrate residues attached to Asn227, and its molecular mass was calculated to be 28,690 Da. The sequence comparison with ricin A-chain shows that 33% of the residues of momordin-a are identical to those of ricin A-chain and that the residues involved in the catalytic site of the ricin A-chain are conserved in momordin-a.

Amino Acid Sequence↗

PubMed1993

Molecular analysis of ependymins from the cerebrospinal fluid of the orders Clupeiformes and Salmoniformes: no indication for the existence of an euteleost infradivision.

Ependymins represent the predominant protein constituents in the cerebrospinal fluid of many teleost fish and they are synthesized in meningeal fibroblasts. Here, we present the ependymin sequences from the herring (Clupea harengus) and the pike (Esox lucius). A comparison of ependymin homologous sequences from three different orders of teleost fish (Salmoniformes, Cypriniformes, and Clupeiformes) revealed the highest similarity between Clupeiformes and Cypriniformes. This result is unexpected because it does not reflect current systematics, in which Clupeiformes belong to a separate infradivision (Clupeomorpha) than Salmoniformes and Cypriniformes (Euteleostei). Furthermore, in Salmoniformes the evolutionary rate of ependymins seems to be accelerated mainly on the protein level. However, considering these inconstant rates, neither neighbor-joining trees nor DNA parsimony methods gave any indication that a separate euteleost infradivision exists.

Amino Acid Sequence↗

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↗

PubMed1998

Microbial gene identification using interpolated Markov models.

This paper describes a new system, GLIMMER, for finding genes in microbial genomes. In a series of tests on Haemophilus influenzae , Helicobacter pylori and other complete microbial genomes, this system has proven to be very accurate at locating virtually all the genes in these sequences, outperforming previous methods. A conservative estimate based on experiments on H.pylori and H. influenzae is that the system finds >97% of all genes. GLIMMER uses interpolated Markov models (IMMs) as a framework for capturing dependencies between nearby nucleotides in a DNA sequence. An IMM-based method makes predictions based on a variable context; i.e., a variable-length oligomer in a DNA sequence. The context used by GLIMMER changes depending on the local composition of the sequence. As a result, GLIMMER is more flexible and more powerful than fixed-order Markov methods, which have previously been the primary content-based technique for finding genes in microbial DNA.

Algorithms↗