Molecular Sequence Alignment Research Articles

Phylogenetic network-assisted rooting of unrooted gene trees

Gene trees inferred from molecular sequence alignments are typically unrooted, and determining the most credible rooting edge is a classical problem in computational biology. One approach to solve this problem is unrooted reconciliation, where the rooting edge is postulated based on the split of the root from a given species tree. In this paper, we propose a novel variant of the gene tree rooting problem, where the gene tree root is inferred using a phylogenetic network of the species present in the gene tree. To obtain the best rooting, unrooted reconciliation can be applied, where the unrooted gene tree is jointly reconciled with a set of splits inferred from the network. However, the exponential size of the set induced by display trees of the network makes this approach computationally prohibitive. To address this, we propose a broader and easier-to-control set of splits based on the structural properties of the network. We then derive exact mathematical formulas for the rooting problem and propose two general rooting algorithms to handle cases where the input network does not meet the initial requirements. Our experimental study based on simulated gene trees and networks demonstrates that our algorithms infer gene tree rootings correctly or with a small error in most cases.

Biosynthesis of 3-Hydroxyphloretin Using Rational Design of 4-Hydroxyphenylacetate 3-Monooxygenase.

The compound 3-hydroxyphloretin is a typical dihydrochalcone that can be obtained in plants by the 3-hydroxylation of phloretin. Here, the flavin-dependent two-component monooxygenase (HpaBC) derived from Pseudomonas aeruginosa was used to convert phloretin into 3-hydroxyphloretin. Following molecular docking and sequence alignment, modifications to the substrate pocket and loop of PaHpaBC were rationally designed, and mutant residues were selected. The results showed that the mutant Q212G/F292A/Q376N gave the best yield of 3-hydroxyphloretin and showed improved catalytic efficiency. Under optimal reaction condition, 2.03 g/L of 3-hydroxyphloretin was produced in the whole-cell catalysis experiment. Molecular docking and molecular dynamics simulations were used to analyze mutants and elucidate the potential mechanism. It was found that the increase in 3-hydroxyphloretin yield was due to the improvement in the flexibility of the loop and the expansion of its substrate pocket. This strategy based on loop and substrate pocket modification has significance in the engineering of PaHpaB.

Genome-Wide Identification of BrCAX Genes and Functional Analysis of BrCAX1 Involved in Ca2+ Transport and Ca2+ Deficiency-Induced Tip-Burn in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Calcium (Ca2+) plays essential roles in plant growth and development. Ca2+ deficiency causes a physiological disorder of tip-burn in Brassiceae crops and is involved in the regulation of cellular Ca2+ homeostasis. Although the functions of Ca2+/H+ exchanger antiporters (CAXs) in mediating transmembrane transport of Ca2+ have been extensively characterized in multiple plant species, the potential roles of BrCAX genes remain unclear in Chinese cabbage. In this study, eight genes of the BrCAX family were genome-widely identified in Chinese cabbage. These BrCAX proteins contained conserved Na_Ca_ex domain and belonged to five members of the CAX family. Molecular evolutionary analysis and sequence alignment revealed the evolutionary conservation of BrCAX family genes. Expression profiling demonstrated that eight BrCAX genes exhibited differential expression in different tissues and under heat stress. Furthermore, Ca2+ deficiency treatment induced the typical symptoms of tip-burn in Chinese cabbage seedlings and a significant decrease in total Ca2+ content in both roots and leaves. The expression changes in BrCAX genes were related to the response to Ca2+ deficiency-induced tip-burn of Chinese cabbage. Specially, BrCAX1-1 and BrCAX1-2 genes were highly expressed gene members of the BrCAX family in the leaves and were significantly differentially expressed under Ca2+ deficiency stress. Moreover, overexpression of BrCAX1-1 and BrCAX1-2 genes in yeast and Chinese cabbage cotyledons exhibited a higher Ca2+ tolerance, indicating the Ca2+ transport capacity of BrCAX1-1 and BrCAX1-2. In addition, suppression expression of BrCAX1-1 and BrCAX1-2 genes reduced cytosolic Ca2+ levels in the root tips of Chinese cabbage. These results provide references for functional studies of BrCAX genes and to investigate the regulatory mechanisms underlying Ca2+ deficiency disorder in Brassiceae vegetables.

Characterization of a GH50 β-Agarase: A Biotechnological Tool for Preparing Oligosaccharides from Agarose and Porphyran.

Agarase is of vital significance for functional agaro-oligosaccharides production from algal dived agarose. Especially, the exolytic agarases have the advantage of obtaining agaro-oligosaccharides with a specific degree of polymerization. Herein, we cloned and expressed a novel glycoside hydrolase (GH) 50 family β-agarase OUC-PgJC50 from Photobacterium gaetbulicola. The degradation pattern analysis indicated that OUC-PgJC50 not only showed an exolytic activity with main products of neoagarotetraose from hydrolyzing agarose but also show a hydrolytic activity to transform neoagarotetraose into neoagarobiose. This is the first time that the discovery of a neoagarotetraose-producing exolytic GH50 β-agarase possesses the activity to transform neoagarotetraose into neoagarobiose, which provided new insight into the recognition of the degradation mode of agarases. Molecular docking and sequence alignment analysis further revealed the His654 residue in OUC-PgJC50 may play a vital role in forming a strong force with l-AHG residue at -4 subsite that helps to produce neoagarotetraose from catalyzing agarose. Moreover, the catalytic ability of OUC-PgJC50 toward another agar polysaccharide porphyran was also described that could hydrolyze porphyran into sulfated oligosaccharides, in which the LA6S-d-Gal was the main products. This study is of vital significance for developing the application range of GH50 β-agarases.

An effective computational-screening strategy for simultaneously improving both catalytic activity and thermostability of α-l-rhamnosidase.

Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double-screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α-l-rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, A355N, S356Y, and D525N among the seven mutant candidates were predicted with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve root mean squared deviation, electrostatic, Van der Waal interaction, and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes.

Utilization of computational techniques and tools to introduce or reinforce knowledge of biochemistry and protein structure–function relationships

Utilization of computational techniques and tools to introduce or reinforce knowledge of biochemistry and protein <scp>structure–function</scp> relationships

SPAligner: alignment of long diverged molecular sequences to assembly graphs

BackgroundGraph-based representation of genome assemblies has been recently used in different contexts — from improved reconstruction of plasmid sequences and refined analysis of metagenomic data to read error correction and reference-free haplotype reconstruction. While many of these applications heavily utilize the alignment of long nucleotide sequences to assembly graphs, first general-purpose software tools for finding such alignments have been released only recently and their deficiencies and limitations are yet to be discovered. Moreover, existing tools can not perform alignment of amino acid sequences, which could prove useful in various contexts — in particular the analysis of metagenomic sequencing data.ResultsIn this work we present a novel SPAligner (Saint-Petersburg Aligner) tool for aligning long diverged nucleotide and amino acid sequences to assembly graphs. We demonstrate that SPAligner is an efficient solution for mapping third generation sequencing reads onto assembly graphs of various complexity and also show how it can facilitate the identification of known genes in complex metagenomic datasets.ConclusionsOur work will facilitate accelerating the development of graph-based approaches in solving sequence to genome assembly alignment problem. SPAligner is implemented as a part of SPAdes tools library and is available on Github.

Markov-Modulated Continuous-Time Markov Chains to Identify Site- and Branch-Specific Evolutionary Variation in BEAST

Markov models of character substitution on phylogenies form the foundation of phylogenetic inference frameworks. Early models made the simplifying assumption that the substitution process is homogeneous over time and across sites in the molecular sequence alignment. While standard practice adopts extensions that accommodate heterogeneity of substitution rates across sites, heterogeneity in the process over time in a site-specific manner remains frequently overlooked. This is problematic, as evolutionary processes that act at the molecular level are highly variable, subjecting different sites to different selective constraints over time, impacting their substitution behavior. We propose incorporating time variability through Markov-modulated models (MMMs), which extend covarion-like models and allow the substitution process (including relative character exchange rates as well as the overall substitution rate) at individual sites to vary across lineages. We implement a general MMM framework in BEAST, a popular Bayesian phylogenetic inference software package, allowing researchers to compose a wide range of MMMs through flexible XML specification. Using examples from bacterial, viral, and plastid genome evolution, we show that MMMs impact phylogenetic tree estimation and can substantially improve model fit compared to standard substitution models. Through simulations, we show that marginal likelihood estimation accurately identifies the generative model and does not systematically prefer the more parameter-rich MMMs. To mitigate the increased computational demands associated with MMMs, our implementation exploits recent developments in BEAGLE, a high-performance computational library for phylogenetic inference. [Bayesian inference; BEAGLE; BEAST; covarion, heterotachy; Markov-modulated models; phylogenetics.]

Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes α-phenylalanine to β-phenylalanine, and could be used to synthesize unnatural β-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pam), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, 15N-(2S)-α-phenylalanine, (2S)-(3-2H2)-α-phenylalanine and (2S,3S)-[2,3-2H2]-α-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of α-phenylalanine. The PaPAM shuttles the α-NH2 of α-phenylalanine to β site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to α site to produce β-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.

Scientific Discovery Games for Biomedical Research.

Over the past decade, scientific discovery games (SDGs) have emerged as a viable approach for biomedical research, engaging hundreds of thousands of volunteer players and resulting in numerous scientific publications. After describing the origins of this novel research approach, we review the scientific output of SDGs across molecular modeling, sequence alignment, neuroscience, pathology, cellular biology, genomics, and human cognition. We find compelling results and technical innovations arising in problem-oriented games such as Foldit and Eterna and in data-oriented games such as EyeWire and Project Discovery. We discuss emergent properties of player communities shared across different projects, including the diversity of communities and the extraordinary contributions of some volunteers, such as paper writing. Finally, we highlight connections to artificial intelligence, biological cloud laboratories, new game genres, science education, and open science that may drive the next generation of SDGs.

Pathophysiology, Etiology, Epidemiology of Type 1 Diabetes and Computational Approaches for Immune Targets and Therapy.

Autoimmune diseases occur when the body's natural defense system fails to differentiate its own cells from the foreign cells and mistakenly attacks the healthy cells. Among the autoimmune diseases, the most common serious disease is the type 1 diabetes (T1D). Biomarkers like c-peptide, autoantibodies, and glycated molecules are now widely used for the early diagnosis of diabetes. However, the diverse nature of biomarkers and the available autoantibodies as biomarkers are not enough to differentiate the heterogeneity inherent in T1D. Novel biomarkers have allowed the introduction of bioinformatics for assimilating the new data into clinical tools. Computer-aided drug design contributes to the discovery of novel autoantibodies, and molecular docking promises to enhance it. Moreover, the study of the pathophysiology of diabetes via molecular simulation has been proposed. In this review article, we focus on the characterization of the etiology, epidemiological factors, and mechanisms of hyperglycemia that induce cellular damage due to oxidative stress and proinflammatory responses. We also decribe novel biomarkers used for the detection of β-cell destruction and diagnosis at early stages. Bioinformatics tools including molecular docking, sequence alignment, and homology modeling are also presented. This report supports researchers in drug design, in disease detection at an early phase, and in therapy development for T1D-associated complications.

Sequence Similarity: An inquiry based and "under the hood" approach for incorporating molecular sequence alignment in introductory undergraduate biology courses

Sequence Similarity: An inquiry based and "under the hood" approach for incorporating molecular sequence alignment in introductory undergraduate biology courses

Clinical application of high mobility group box nuclear protein 1 subcomponent in the progression of liver cancer

The biological functions of high-mobility group (HMG) proteins include regulation of DNA replication, transcription, recombination and repair. According to molecular weight, sequence alignment and DNA structural characteristics, HMG proteins are subdivided into three superfamilies (HMGA, HMGB and HMGN). Recently, HMGB family members (HMGB1, HMGB2, HMGB3, and HMGB4) found to interact with hepatitis B or C virus. Therefore, activation of relevant signaling molecules to regulate transcription of genes related to hepatocellular carcinoma as a mediator of inflammation promoting HCC progression has attracted considerable attentions. This article focuses on the clinical application of the expression of HMGB family members in the process of HCC progression.

Alv: a console-based viewer for molecular sequence alignments

alv: a console-based viewer for molecular sequence alignments

BpWrapper: BioPerl-based sequence and tree utilities for rapid prototyping of bioinformatics pipelines

BackgroundAutomated bioinformatics workflows are more robust, easier to maintain, and results more reproducible when built with command-line utilities than with custom-coded scripts. Command-line utilities further benefit by relieving bioinformatics developers to learn the use of, or to interact directly with, biological software libraries. There is however a lack of command-line utilities that leverage popular Open Source biological software toolkits such as BioPerl (http://bioperl.org) to make many of the well-designed, robust, and routinely used biological classes available for a wider base of end users.ResultsDesigned as standard utilities for UNIX-family operating systems, BpWrapper makes functionality of some of the most popular BioPerl modules readily accessible on the command line to novice as well as to experienced bioinformatics practitioners. The initial release of BpWrapper includes four utilities with concise command-line user interfaces, bioseq, bioaln, biotree, and biopop, specialized for manipulation of molecular sequences, sequence alignments, phylogenetic trees, and DNA polymorphisms, respectively. Over a hundred methods are currently available as command-line options and new methods are easily incorporated. Performance of BpWrapper utilities lags that of precompiled utilities while equivalent to that of other utilities based on BioPerl. BpWrapper has been tested on BioPerl Release 1.6, Perl versions 5.10.1 to 5.25.10, and operating systems including Apple macOS, Microsoft Windows, and GNU/Linux. Release code is available from the Comprehensive Perl Archive Network (CPAN) at https://metacpan.org/pod/Bio::BPWrapper. Source code is available on GitHub at https://github.com/bioperl/p5-bpwrapper.ConclusionsBpWrapper improves on existing sequence utilities by following the design principles of Unix text utilities such including a concise user interface, extensive command-line options, and standard input/output for serialized operations. Further, dozens of novel methods for manipulation of sequences, alignments, and phylogenetic trees, unavailable in existing utilities (e.g., EMBOSS, Newick Utilities, and FAST), are provided. Bioinformaticians should find BpWrapper useful for rapid prototyping of workflows on the command-line without creating custom scripts for comparative genomics and other bioinformatics applications.

Introgression of bacterial wilt resistance from Solanum melongena to S . t uberosum through asymmetric protoplast fusion

Bacterial wilt (BW) caused by Ralstonia solanacearum is an important disease of many plant species especially Solanaceae. To compensate for lack of BW resistance in cultivated potato, we fused UV-treated protoplasts of a resistant eggplant variety with protoplasts of a susceptible potato clone to obtain 32 somatic hybrids. Although asymmetric protoplast fusion has the potential to transfer traits from distant species, introgression frequency and preference of alien fragments remain obscure, as well as the genetic basis for control of a trait. In the present research, the genome components of 32 somatic hybrids were determined by parent-specific SSRs. Each hybrid had integrated from one to eight alien chromosome fragments, providing a foundation for selection of BW resistance transmitted from eggplant. When the selected eggplant sequences were aligned with potato genome sequence it showed a similarity of 46.7 %, suggesting a large genetic distance between these two species. The results also revealed that introgression of eggplant fragments is non-selective, which may allow any part of alien chromosomes to be integrated. Distribution of eggplant loci in individual hybrids suggested a possible relationship between markers emk03O04, emi04P17 and emd13E02a and BW resistance, which are potential loci that control target traits and therefore deserve further investigation. With genome-wide selection of parent-specific molecular markers and sequence alignment, the present research substantiated interspecific introgression of a trait lacking in potato. Moreover, an efficient strategy was established to estimate genomic components of the somatic hybrids, and to explore the candidate loci that associate with target traits.

Aligning two molecular sequences using genetic operators in grey wolf optimiser technique

Sequence analysis is one of the most important concepts in the domain of bioinformatics. Molecular sequence alignment is a predominant problem in sequence analysis. In this paper, we proposed a new approach for pairwise sequence alignment using a recent meta-heuristic algorithm called Grey Wolf Optimiser GWO technique in which genetic operators are integrated to yield efficient solutions. This algorithm obtains the initial set of alignments by inserting gaps randomly and uses the search agents in GWO for exploration and exploitation. In addition to this, genetic operators like crossover and mutation are applied for faster convergence. A novel horizontal crossover and a single-point crossover that suits, particularly for sequence alignment problem, are employed in this paper. Here, two mutations are used depending upon their threshold value. This threshold value depends on a novel fitness function F

Classification of molecular sequence data using Bayesian phylogenetic mixture models

Rate variation among the sites of a molecular sequence is commonly found in applications of phylogenetic inference. Several approaches exist to account for this feature but they do not usually enable the investigator to pinpoint the sites that evolve under one or another rate of evolution in a straightforward manner. The focus is on Bayesian phylogenetic mixture models, augmented with allocation variables, as tools for site classification and quantification of classification uncertainty. The method does not rely on prior knowledge of site membership to classes or even the number of classes. Furthermore, it does not require correlated sites to be next to one another in the sequence alignment, unlike some phylogenetic hidden Markov or change-point models. In the approach presented, model selection on the number and type of mixture components is conducted ahead of both model estimation and site classification; the steppingstone sampler (SS) is used to select amongst competing mixture models. Example applications of simulated data and mitochondrial DNA of primates illustrate site classification via ‘augmented’ Bayesian phylogenetic mixtures. In both examples, all mixtures outperform commonly-used models of among-site rate variation and models that do not account for rate heterogeneity. The examples further demonstrate how site classification is readily available from the analysis output. The method is directly relevant to the choice of partitions in Bayesian phylogenetics, and its application may lead to the discovery of structure not otherwise recognised in a molecular sequence alignment. Computational aspects of Bayesian phylogenetic model estimation are discussed, including the use of simple Markov chain Monte Carlo (MCMC) moves that mix efficiently without tempering the chains. The contribution to the field of Bayesian phylogenetics is in (1) the use of mixture models augmented with allocation variables as tools for site classification and quantification of classification uncertainty, (2) the successful application of SS for selection of phylogenetic mixtures, and (3) the development of novel MCMC aspects of relevance to Bayesian phylogenetic models—whether mixtures or not.11The MCMC methods discussed in this paper have been coded in a C program; source files are available upon request. Supplementary material is available online (see Appendix A).

Characterization and site-directed mutagenesis of an α-galactosidase from the deep-sea bacterium Bacillus megaterium

A novel gene (BmelA) (1323bp) encoding an α-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35°C and pH 8.5–9.0 in 50mM Tris–HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-d-galactopyranoside (pNP-α-Gal), raffinose and stachyose being 1.02±0.02, 2.24±0.11 and 3.42±0.17mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (kcat/Km) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted “tunnel” structure and NAD+ binding to the enzyme.

Population Genetic Structure of red mullet (Mullus barbatus L.) in Turkish Sea Based on Mitochondrial DNA

Aim: Mullus barbatus (red mullet) is a commercial fish species naturally distributed from Eastern Atlantic: British Isles to Dakar, Senegal, Canary Islands, Mediterranean and Black Sea. There is no study in our knowledge aimed to determine population genetic structuring and genetic stocks of M. barbatus species in territorial waters of Turkey. Only a few studies have been carried out on their genetics in Turkey which are limited to determination of phylogenetic relationships between species in familia of Mullidae. In this study population genetic structure and genetic diversity of red mullet (Mullus barbatus L.) in Turkish Seas was determined using sequence data of mitochondrial DNA control region.