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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↗
PubMed1996

Secondary structure prediction and unrefined tertiary structure prediction for cyclin A, B, and D.

We present heuristic-based predictions of the secondary and tertiary structures of cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a beta-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure.

Amino Acid Sequence↗
PubMed1994

Peptide sequences binding to MHC class I proteins.

Motifs for peptides which bind specifically to the human class I major histocompatibility complex molecules HLA-A2 and B7 were determined by sequence analysis of class I-bound peptides selected from a random synthetic library of nonamers. Thirteen individual peptides were sequenced for HLA-A2, twelve individual and nine pooled peptides were sequenced for HLA-B7. Analysis of sequence alignment implicated four peptide positions in potential contact with the class I HLA-A2 molecule and three positions for the HLA-B7 molecule. The results demonstrate that a synthetic peptide library can be used to identify allele-specific motifs for class I molecules, providing information comparable to the results obtained from sequencing endogenous peptides. This method utilizes denatured class I heavy chains, and similar results were obtained using a class I protein purified from mammalian cells or by expression in Escherichia coli. This method has the potential to detect peptides which may not be generated physiologically, but due to their binding properties, may be valuable to predict or engineer immunomodulatory T cell epitopes.

Amino Acid Sequence↗
PubMed1994

Reconstructing evolutionary trees from DNA and protein sequences: paralinear distances.

The reconstruction of phylogenetic trees from DNA and protein sequences is confounded by unequal rate effects. These effects can group rapidly evolving taxa with other rapidly evolving taxa, whether or not they are genealogically related. All algorithms are sensitive to these effects whenever the assumptions on which they are based are not met. The algorithm presented here, called paralinear distances, is valid for a much broader class of substitution processes than previous algorithms and is accordingly less affected by unequal rate effects. It may be used with all nucleic acid, protein, or other sequences, provided that their evolution may be modeled as a succession of Markov processes. The properties of the method have been proven both analytically and by computer simulations. Like all other methods, paralinear distances can fail when sequences are misaligned or when site-to-site sequence variation of rates is extensive. To examine the usefulness of paralinear distances, the "origin of the eukaryotes" has been investigated by the analysis of elongation factor Tu sequences with a variety of sequence alignments. It has been found that the order in which sequences are pairwise aligned strongly determines the topology which is reconstructed by paralinear distances (as it does for all other reconstruction methods tested). When the parts of the alignment that are unaffected by alignment order are analyzed, paralinear distances strongly select the eocyte topology. This provides evidence that the eocyte prokaryotes are the closest prokaryotic relatives of the eukaryotes.

Algorithms↗
PubMed1984

A structural model of human erythrocyte protein 4.1.

Limited proteolysis and specific chemical cleavage methods have enabled a detailed structural characterization of human erythrocyte protein 4.1. This protein is composed of two chemically very similar polypeptide chains (a and b) with apparent molecular masses of 80,000 and 78,000 daltons. Cleavage of protein 4.1 at cysteine residues by 2-nitro-5-thiocyanobenzoic acid produces a series of doublets which differ by approximately 2,000 daltons and have identical peptide maps. Alignment of these peptides by mapping analysis has localized 4 cysteine residues within a 17,000-dalton segment on both a and b polypeptides. Mild chymotryptic treatment at 0 degrees C cleaves protein 4.1 primarily in three central locations and generates two families of unrelated peptides. Analysis of these fragments in two-dimensional gels and by peptide mapping reveals an unusual polarity in protein 4.1 structure in that each polypeptide chain contains two segments, one relatively acidic the other basic, that are segregated at opposite ends of the molecule. The basic region is digested into a cysteine-rich 30,000-dalton domain which resists further breakdown while the acidic region is readily degraded into smaller fragments. The peptides derived from the acidic region all appear as doublets suggesting that protein 4.1 a and b polypeptides differ close to the terminus of the acidic end. Similar phosphorylation sites occur on both polypeptides within a segment some 24,000-34,000 daltons from the acidic terminus.

Blood Proteins↗
PubMed1984

Conformation and packing of unsaturated chains in crystals of cholesteryl nervonate at 123 K.

At 123 K, cholesterol cis-15-tetracosenoate (cholesteryl nervonate, C51H90O2) is monoclinic, space group P2(1) with a = 12.948(5), b = 8.805(5), c = 42.98(5) A, beta = 105.93(3) degrees, [lambda(CuK alpha) = 1.5418 A], having two independent molecules (A) and (B) in the unit cell. The crystal structure at 123 K has been determined from 6329 reflections with sin theta/lambda less than 0.54 A-1, of which 1990 gave I greater than 2 sigma(I). Structure refinement by Fourier methods and block diagonal least squares gave R = 0.232 for all reflections, R = 0.135 for those with I greater than 2 sigma(I). The crystal structure consists of layers in which there is close packing of cholesteryl groups and the proximal segments of the ester chains. This layer structure occurs also in cholesteryl palmitoleate and the alkanoate esters C9 through C12. Because the nervonate chains are longer, they become aligned in the interface region between layers, but without a regular subcell structure being established. The nervonate (A)-chain is almost extended, while the (B)-chain has two bends, one in the saturated region. Both chains have complex dislocations at the cis-double bond.

Cholesterol Esters↗
PubMed1983

[The primary structure of hemoglobins of the rock hyrax (Procavia habessinica, Hyracoidea): insertion of glutamine in the alpha chains].

The chromatography of the hemoglobin of the rock hyrax (Procavia habessinica) gives two components (73% HbI and 27% HbII). The amino-acid analysis and the sequences of the globin chains elucidated with the phenylthiohydantoin method, did not show any differences between the alpha I and alpha II or beta I and beta II chains, respectively. The different chromatographical behaviour cannot be explained. After chain separation by chromatography on CM-52 cellulose, all four primary structures were elucidated automatically in a sequenator on the chains and the tryptic peptides. In 20% of the beta I chains the N-terminal valine was blocked by acetyl. The alignment was performed by homology with the chains of human adult hemoglobin. The alpha chain of the rock hyrax has 142 amino-acid residues, i.e. one residue more than normal mammalian alpha chains, caused by an insertion of glutamine in the GH region supposed between positions 115 and 116. A comparison of human and hyrax hemoglobins shows an exchange of 21 amino-acid residues in the alpha chains and of 24 in the beta chains. Some substitutions in alpha 1 beta 1 contacts and in the surrounding of the heme are not supposed to effect the function of the hemoglobin. The phylogenetic relationship between the rock hyrax and the Indian elephant (Elephas maximus) on the one hand and with some Perissodactyla on the other, is discussed. Up to now the exchanges of alpha 110(G17)Ala leads to Ser and beta 56(D7)Gly leads to His have only been found in hyrax and elephant. This indicates a certain relationship between Hyracoidea and Proboscidea.

Amino Acid Sequence↗
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↗
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↗