Patterns Of Amino Acid Replacement Research Articles

Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Analysis for the Direct Detection of SARS-CoV-2 in Nasopharyngeal Swabs

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 106.7 viral copies. This value equates to 107.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.

A transcriptomic approach to ribbon worm systematics (nemertea): resolving the pilidiophora problem.

Resolving the deep relationships of ancient animal lineages has proven difficult using standard Sanger-sequencing approaches with a handful of markers. We thus reassess the relatively well-studied phylogeny of the phylum Nemertea (ribbon worms)-for which the targeted gene approaches had resolved many clades but had left key phylogenetic gaps-by using a phylogenomic approach using Illumina-based de novo assembled transcriptomes and automatic orthology prediction methods. The analysis of a concatenated data set of 2,779 genes (411,138 amino acids) with about 78% gene occupancy and a reduced version with 95% gene occupancy, under evolutionary models accounting or not for site-specific amino acid replacement patterns results in a well-supported phylogeny that recovers all major accepted nemertean clades with the monophyly of Heteronemertea, Hoplonemertea, Monostilifera, being well supported. Significantly, all the ambiguous patterns inferred from Sanger-based approaches were resolved, namely the monophyly of Palaeonemertea and Pilidiophora. By testing for possible conflict in the analyzed supermatrix, we observed that concatenation was the best solution, and the results of the analyses should settle prior debates on nemertean phylogeny. The study highlights the importance, feasibility, and completeness of Illumina-based phylogenomic data matrices.

Reconstructing the phylogenetic history of long-term effective population size and life-history traits using patterns of amino acid replacement in mitochondrial genomes of mammals and birds.

The nearly neutral theory, which proposes that most mutations are deleterious or close to neutral, predicts that the ratio of nonsynonymous over synonymous substitution rates (dN/dS), and potentially also the ratio of radical over conservative amino acid replacement rates (Kr/Kc), are negatively correlated with effective population size. Previous empirical tests, using life-history traits (LHT) such as body-size or generation-time as proxies for population size, have been consistent with these predictions. This suggests that large-scale phylogenetic reconstructions of dN/dS or Kr/Kc might reveal interesting macroevolutionary patterns in the variation in effective population size among lineages. In this work, we further develop an integrative probabilistic framework for phylogenetic covariance analysis introduced previously, so as to estimate the correlation patterns between dN/dS, Kr/Kc, and three LHT, in mitochondrial genomes of birds and mammals. Kr/Kc displays stronger and more stable correlations with LHT than does dN/dS, which we interpret as a greater robustness of Kr/Kc, compared with dN/dS, the latter being confounded by the high saturation of the synonymous substitution rate in mitochondrial genomes. The correlation of Kr/Kc with LHT was robust when controlling for the potentially confounding effects of nucleotide compositional variation between taxa. The positive correlation of the mitochondrial Kr/Kc with LHT is compatible with previous reports, and with a nearly neutral interpretation, although alternative explanations are also possible. The Kr/Kc model was finally used for reconstructing life-history evolution in birds and mammals. This analysis suggests a fairly large-bodied ancestor in both groups. In birds, life-history evolution seems to have occurred mainly through size reduction in Neoavian birds, whereas in placental mammals, body mass evolution shows disparate trends across subclades. Altogether, our work represents a further step toward a more comprehensive phylogenetic reconstruction of the evolution of life-history and of the population-genetics environment.

Genetic characterization and phylogenetic analysis of skunk-associated rabies viruses in North America with special emphasis on the central plains

Across North America the skunk acts as a reservoir for several rabies virus variants. Some of these variants are geographically restricted in range as is the case for the California skunk variant and two distinct variants present in Mexico. In contrast the North Central and South Central skunk rabies viruses are dispersed in overlapping ranges over large areas of the Midwestern region of the United States with the former extending into southern parts of the Canadian prairies. Despite this extensive range, there has been only very limited molecular characterization of these two viral variants. This study has examined the genetic diversity of the rabies viruses associated with North American skunks, with particular emphasis on the South Central skunk variant which was found to comprise three distinct geographically restricted groups of viruses that could in some cases be further sub-divided. The phylogenetic relationships of these groups and sub-groups allowed us to infer the likely direction of spread of these variants in some instances. Patterns of amino acid replacement of North American skunk-associated rabies viruses for both the nucleoprotein and glycoprotein products are also examined. These patterns reflect the virus phylogeny but no amino acid residues associated specifically with the skunk host were identified.

Molecular evolution of cyclin proteins in animals and fungi.

BackgroundThe passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi.ResultsWe constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution.ConclusionsThe results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events.

Hantavirus Evolution in Relation to Its Rodent and Insectivore Hosts: No Evidence for Codivergence

Hantaviruses are considered one of the best examples of a long-term association between RNA viruses and their hosts. Based on the appearance of strong host specificity, it has been suggested that hantaviruses cospeciated with the rodents and insectivores they infect since these mammals last shared a common ancestor, approximately 100 million years ago. We tested this hypothesis of host-virus codivergence in two ways: 1) we used cophylogenetic reconciliation analysis to assess the fit of the virus tree onto that of the host and 2) we estimated the evolutionary rates and divergence times for the Hantavirus genus using a Bayesian Markov Chain Monte Carlo method and similarly compared these with those of their hosts. Our reconciliation analysis provided no evidence for a history of codivergence between hantaviruses and their hosts. Further, the divergence times for the Hantavirus genus were many orders of magnitude too recent to correspond with the timescale of their hosts' speciation. We therefore propose that apparent similarities between the phylogenies of hantaviruses and their mammalian hosts are the result of a more recent history of preferential host switching and local adaptation. Based on the presence of clade-defining amino acids in all genomic segments, we propose that the patterns of amino acid replacement in these viruses are also compatible with a history of host-specific adaptation.

Empirical profile mixture models for phylogenetic reconstruction

Previous studies have shown that accounting for site-specific amino acid replacement patterns using mixtures of stationary probability profiles offers a promising approach for improving the robustness of phylogenetic reconstructions in the presence of saturation. However, such profile mixture models were introduced only in a Bayesian context, and are not yet available in a maximum likelihood (ML) framework. In addition, these mixture models only perform well on large alignments, from which they can reliably learn the shapes of profiles, and their associated weights. In this work, we introduce an expectation-maximization algorithm for estimating amino acid profile mixtures from alignment databases. We apply it, learning on the HSSP database, and observe that a set of 20 profiles is enough to provide a better statistical fit than currently available empirical matrices (WAG, JTT), in particular on saturated data.

Molecular characterization of major histocompatibility complex class I genes from the giant panda (Ailuropoda melanoleuca)

The major histocompatibility complex class I genes play crucial roles in the adaptive immune system of vertebrates against intracellular pathogens. To date, no class I genes from the giant panda (Ailuropoda melanoleuca) has been reported, even none from species of Ursidae. In this study, we successfully identified three class I genes from a giant panda bacterial artificial chromosome library and designated them as Aime-128, 152, and 1906, respectively. Pairwise sequence alignments revealed that (1) the Aime-1906 always possessed the lowest identities (52-86%) in different regions compared with the Aime-128 and 152 and (2) the Aime-128 also varied from the Aime-152 in the regions of 5' untranslated region (UTR), 3' UTR, and exon1, whose similarities were 83%, 87%, and 91%, respectively. Comparison of structure characteristics indicated that the Aime-128 possessed all conserved amino acids important to the function of antigen presentation while the Aime-152 and 1906 presented two and five mutated residues. Analysis of phylogenetic trees demonstrated that the Aime-128, 152, and 1906 were clustered into three different branches with 99% or 100% bootstrap values. As a result, these three kinds of evidence supported that the Aime-1906, 152, and 128 should be derived from different loci. Furthermore, in view of a prestop codon in the exon 7 and patterns of amino acid replacement within alleles, the Aime-1906 gene is predicted to be a nonclassical locus, which is most closely related to dog leukocyte antigen 79 in the phylogenetic tree constructed with various mammalian class I loci.

Improvement of molecular phylogenetic inference and the phylogeny of Bilateria

Inferring the relationships among Bilateria has been an active and controversial research area since Haeckel. The lack of a sufficient number of phylogenetically reliable characters was the main limitation of traditional phylogenies based on morphology. With the advent of molecular data, this problem has been replaced by another one, statistical inconsistency, which stems from an erroneous interpretation of convergences induced by multiple changes. The analysis of alignments rich in both genes and species, combined with a probabilistic method (maximum likelihood or Bayesian) using sophisticated models of sequence evolution, should alleviate these two major limitations. We applied this approach to a dataset of 94 genes and 79 species using CAT, a previously developed model accounting for site-specific amino acid replacement patterns. The resulting tree is in good agreement with current knowledge: the monophyly of most major groups (e.g. Chordata, Arthropoda, Lophotrochozoa, Ecdysozoa, Protostomia) was recovered with high support. Two results are surprising and are discussed in an evo-devo framework: the sister-group relationship of Platyhelminthes and Annelida to the exclusion of Mollusca, contradicting the Neotrochozoa hypothesis, and, with a lower statistical support, the paraphyly of Deuterostomia. These results, in particular the status of deuterostomes, need further confirmation, both through increased taxonomic sampling, and future improvements of probabilistic models.

Thromboxane synthase (TBXAS1) polymorphisms in African-American and Caucasian populations: evidence for selective pressure

Thromboxane synthase (TBXAS1), a cytochrome P450 enzyme, converts prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and inducer of platelet aggregation. Thromboxane A2 has been implicated in modulating cell cytotoxicity and in tumor growth and metastasis. Twelve coding-region variants were identified in the human TBXAS1 gene in 48 African-American and 46 Caucasian individuals, of which eight were amino-acid substitutions. The latter were confirmed in an independent Caucasian population (n=94 unrelated individuals). We performed an evolutionary analysis of patterns of nucleotide diversity and identified patterns of amino acid replacement in human-mouse comparisons consistent with purifying selection on an inter-species time scale using the McDonald-Kreitman test. We also observed patterns of nucleotide diversity within humans consistent with purifying selection acting on existing polymorphism using Tajima's D within coding regions. These evolutionary tests suggest that some of the rare coding variations observed in the human population are deleterious. We used two sequence-homology-based software programs and molecular modeling to predict the potential impact of these polymorphisms on TBXAS1 function. The c.772C>T (p.Lys258Glu), c.1249C>G (p.Gln417Glu), and c.1348G>A (p.Glu450Lys) substitutions are predicted as most likely to alter protein function; another, c.1352C>A (p.Thr451Asn), may also affect function. Given the evolutionary evidence, these variants may be functional and therefore of relevance for disease endpoints related to inflammation and angiogenesis, as well as for the pharmacogenetics of non-steroidal anti-inflammatory drugs.

Specific protease mutation patterns are associated with oropharyngeal candidiasis in a New Orleans patient cohort of HIV-infected individuals

HIV-1 protease gene sequences were obtained from peripheral blood, saliva or oral tissues of 35 HIV+ patients using nested amplification and manual sequencing of PCR products. Of the 35 HIV+ patients 9 had clinical oropharyngeal candidiasis (OPC) while 26 did not, and only 4 patients were on protease inhibitor (PI) therapy. These patients were collected prior to major use of HAART therapy in New Orleans, Louisiana. Analysis of 172 amino acid sequences revealed unique patterns of mutation that were in most cases independent of the type of cell from which DNA was isolated and were, instead, primarily dependent on the individual patient. Principal component analysis indicated that approximately 50% of the variance of the amino acid replacements could be explained by patterns of change seen in only five patients. Significantly, 4 of these 5 patients were OPC+ indicating that patients with OPC are more likely to express a principal mutation pattern than patients without OPC (p = 0.002, Chi square). Dendrograms revealed that these five patients clustered separately from each other and from HIV-1LAI suggesting that principal mutation patterns as well as OPC are independent of viral evolution. In conclusion, prior to widespread use of PI therapy to combat HIV-1, patients with OPC exhibited unique patterns of amino acid replacements within the HIV-1 protease.

Physicochemical Evolution and Molecular Adaptation of the Cetacean and Artiodactyl Cytochrome b Proteins

Cetaceans have most likely experienced metabolic shifts since evolutionarily diverging from their terrestrial ancestors, shifts that may be reflected in the proteins such as cytochrome b that are responsible for metabolic efficiency. However, accepted statistical methods for detecting molecular adaptation are largely biased against even moderately conservative proteins because the primary criterion involves a comparison of nonsynonymous and synonymous substitution rates (dN/dS); they do not allow for the possibility that adaptation may come in the form of very few amino acid changes. We apply the MM01 model to the possible molecular adaptation of cytochrome b among cetaceans because it does not rely on a dN/dS ratio, instead evaluating positive selection in terms of the amino acid properties that comprise protein phenotypes that selection at the molecular level may act upon. We also apply the codon-degeneracy model (CDM), which focuses on evaluating overall patterns of nucleotide substitution in terms of base exchange, codon position, and synonymy to estimate the overall effect of selection. Using these relatively new models, we characterize the molecular adaptation that has occurred in the cetacean cytochrome b protein by comparing revealed amino acid replacement patterns to those found among artiodactyls, the modern terrestrial mammals found to be most closely related to cetaceans. Our findings suggest that several regions of the cetacean cytochrome b protein have experienced molecular adaptation. Also, these adaptations are spatially associated with domain structure, protein function, and the structure and function of the cytochrome bc(1) complex and its constituents. We also have found a general correlation between the results of the analytical software programs TreeSAAP (which implements the MM01 model) and CDM (which implements the codon-degeneracy model).

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA Thr and tRNA Pro genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.

A branch-and-bound algorithm for the inference of ancestral amino-acid sequences when the replacement rate varies among sites: Application to the evolution of five gene families

We developed an algorithm to reconstruct ancestral sequences, taking into account the rate variation among sites of the protein sequences. Our algorithm maximizes the joint probability of the ancestral sequences, assuming that the rate is gamma distributed among sites. Our algorithm probably finds the global maximum. The use of 'joint' reconstruction is motivated by studies that use the sequences at all the internal nodes in a phylogenetic tree, such as, for instance, the inference of patterns of amino-acid replacement, or tracing the biochemical changes that occurred during the evolution of a given protein family. We give an algorithm that guarantees finding the global maximum. The efficient search method makes our method applicable to datasets with large number sequences. We analyze ancestral sequences of five gene families, exploring the effect of the amount of among-site-rate-variation, and the degree of sequence divergence on the resulting ancestral states. http://evolu3.ism.ac.jp/~tal/ tal@ism.ac.jp

ADH evolution and the phylogenetic footprint.

The evolution of any given protein reflects the interplay between proximal selective forces involving the conservation of protein structure and function and more general populational factors that shape the action and efficiency of natural selection. In an attempt to address that interplay, we have analyzed patterns of amino acid replacement within a well-conserved molecule, alcohol dehydrogenase (ADH), in the Drosophilidae. A sliding window, moved along the protein sequence in order to quantify the extent of change at each amino acid position, reveals heterogeneous amounts of replacement across the molecule when all ADH sequences are analyzed simultaneously. Surprisingly, the replacement profile for ADH differs significantly in the melanogaster, mulleri, and Hawaiian subgroups, reflecting the imprint of the differing evolutionary histories of each of these assemblages on the evolution of this conservative molecule.

Estimation of Evolutionary Distance between Distantly Related Sequences of Amino Acids, Taking Account of Patterns of Amino Acid Replacement)

When amino acid sequences are distantly related-for instance, when their identity is <0.30-it is difficult to estimate their evolutionary distance. A method called the “similarity distance method” (SD method) was developed to obtain maximum-likelihood estimates of evolutionary distance between amino acid sequences, on the basis of a given pattern of amino acid replacement. Computer simulation revealed that, by using the new method, evolutionary distance can be estimated efficiently even when the expected identity between the sequences is as low as 0.14 and the length of the sequences is only 50 amino acid residues. The new method is useful in phylogenetic analyses of distantly related and/or short sequences of amino acids, such as structural or functional domains commonly found in several proteins.