Clustal Research Articles

Analysis of inherited dysfibrinogenemia pedigree associated with a heterozygous and deletion mutantion in the FGG gene

Objective To analyze the phenotype and genotype of inherited dysfibrinogenemia pedigree associated with a novel heterozygous and deletion mutation in the FGG gene, and to investigate its molecular mechanism. Methods The clinical data were collected from the proband found at our hospital and her family members in April 2016.The activity plasma fibrinogen (Fg: C), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) were detected by coagulation method and the antigen plasma fibrinogen (Fg: Ag ), D-Dimer (D-D), fibrinogen degradation products (FDPs) were analyzed by immunoturbidimetry method. All of the exons and exon-intron boundaries of the genes of FGA, FGB and FGG with the fibrinogen(Fg) were amplified by PCR and followed by direct sequencing. And further verification were performed by cloning sequence and non-denatured polyacrylamide gel electrophoresis and silver staining. The conservatism of mutated gene locus were analyzed by ClustalX- 2.1-win. The change of the protein spatial structure and the intermolecular forces with mutation were analyzed by Pymol. Results The Fg: C of the proband was significantly reduced(0.30 g/L) and the Fg: Ag of the proband was normal (2.00 g/L). Their Fg: C were both significantly reduced and the Fg: Ag were both normal (0.42 g/L, 2.09 g/L & 0.47 g/L, 2.42 g/L, respectively), these were found in her mother and grandma. Genetic analysis revealed a novel heterozygous and deletion mutation with c. 944_c.952 delCCTTTGATG in exon 8 of FGG gene in the proband, predicting a heterozygous 289_291delAla, Phe, Asp mutation. The same mutations were carried by her mother and grandma, but her father and grandpa were normal. Homology analysis indicated that the Ala289, Phe290 and Asp291 were maintained highly conservative in homogenous species. Protein model analysis found that the original hydrogen bonds were disappeared when the deletion mutation happened with the Ala289, Phe290 and Asp291. Conclusion The heterozygous and deletion mutation with 289_291delAla, Phe, Asp in the γ chain of fibrinogen were identified that could cause the rearrangement of the Fg molecular space structure and the reduction of the structure stability, so the mutation probably underly the dysfibrinogenemia in this pedigree.(Chin J Lab Med, 2018, 41: 305-311) Key words: Afibrinogenemia; Pedigree; Loss of heterozygosity; Fibrinogen; Mutation, missense

Analysis of snake venom metalloproteinases from Myanmar Russell's viper transcriptome

Snake venom metalloproteinases (SVMPs) are the key enzymes in Russell's viper (RV) venom which target all important components of haemostasis, such as clotting factors, platelets, endothelial cells and basement membrane. The structural diversity of SVMPs contributes to the broad spectrum of biological activities. The aim of the study was to investigate the SVMP transcript profile to gain better insights into the characteristic clinical manifestations of the Myanmar Russell's viper (MRV) bites that distinguish it from the RVs of other habitats. Next generation sequencing (RNA-Seq) of mRNA from MRV venom glands (2 males and 1 female) was performed on an Illumina HiSeq2000 platform and then de novo assembled using Trinity software. A total of 59 SVMP contigs were annotated through a Blastn search against the serpent nucleotide database from NCBI. Among them, disintegrins were the most abundant transcripts (75%) followed by the P-III class SVMPs (25%). The P-II SVMPs were scarce (0.002%), while no P-I SVMPs were detectable in the transcriptome. For detailed structural analysis, contigs were conceptually translated and compared with amino acid sequences from other RVs and other vipers using Clustal Omega. The RTS-disintegrin (jerdostatin homolog) was the most abundant among transcripts corresponding to 5 disintegrin isoforms. From 10 isoforms of SVMPs, RVV-X, and Vipera lebetina apoptosis-inducing protease (VLAIP) homolog, hereby termed Daboia siamensis AIP (DSAIP), were found to be highly expressed. Venom protein analysis using SDS-PAGE followed by mass spectrometry revealed that the disintegrin was scarce, while the latter two SVMPs were abundant. These two proteins can contribute to severe clinical manifestations caused by MRV envenomation.

JABAWS 2.2 distributed web services for Bioinformatics: protein disorder, conservation and RNA secondary structure.

SummaryJABAWS 2.2 is a computational framework that simplifies the deployment of web services for Bioinformatics. In addition to the five multiple sequence alignment (MSA) algorithms in JABAWS 1.0, JABAWS 2.2 includes three additional MSA programs (Clustal Omega, MSAprobs, GLprobs), four protein disorder prediction methods (DisEMBL, IUPred, Ronn, GlobPlot), 18 measures of protein conservation as implemented in AACon, and RNA secondary structure prediction by the RNAalifold program. JABAWS 2.2 can be deployed on a variety of in-house or hosted systems. JABAWS 2.2 web services may be accessed from the Jalview multiple sequence analysis workbench (Version 2.8 and later), as well as directly via the JABAWS command line interface (CLI) client. JABAWS 2.2 can be deployed on a local virtual server as a Virtual Appliance (VA) or simply as a Web Application Archive (WAR) for private use. Improvements in JABAWS 2.2 also include simplified installation and a range of utility tools for usage statistics collection, and web services querying and monitoring. The JABAWS CLI client has been updated to support all the new services and allow integration of JABAWS 2.2 services into conventional scripts. A public JABAWS 2 server has been in production since December 2011 and served over 800 000 analyses for users worldwide.Availability and implementationJABAWS 2.2 is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws.

Specific primer design of mitochondrial 12S rRNA for species identification in raw meats

Polymerase chain reaction (PCR) is a molecular technique that widely used in agriculture area including species identification in animal-based products for halalness and food safety reasons. Amplification of DNA using PCR needs a primer pair (forward and reverse primers) to isolate specific DNA fragment in the genome. This objective of this study was to design specific primer from mitochondrial 12S rRNA region for species identification in raw beef, pork and chicken meat. Three published sequences, HQ184045, JN601075, and KT626857, were downloaded from National Center for Biotechnology Information (NCBI) website. Furthermore, those reference sequences were used to design specific primer for bovine, pig, and chicken species using primer3 v.0.4.0. A total of 15 primer pairs were picked up from primer3 software. Of these, an universal forward primer and three reverse primers which are specific for bovine, pig, and chicken species were selected to be optimized using multiplex-PCR technique. The selected primers were namely UNI_F (5’-ACC GCG GTC ATA CGA TTA AC-3’), SP_R (5’-AGT GCG TCG GCT ATT GTA GG-3’), BB_R (5’-GAA TTG GCA AGG GTT GGT AA-3’), and A_R (5’-CGG TAT GTA CGT GCC TCA GA-3’). In addition, the PCR products were visualized using 2% agarose gels under the UV light and sequenced to be aligned with reference sequences using Clustal Omega. The result showed that those primers were specifically amplified mitochondrial 12S rRNA regions from bovine, pig, and chicken using PCR. It was indicated by the existence of 155, 357, and 611 bp of DNA bands for bovine, pig, and chicken species, respectively. Moreover, sequence analysis revealed that our sequences were identically similar with reference sequences. It can be concluded that mitochondrial 12S rRNA may be used as a genetic marker for species identification in meat products.

Identification of Protein Biomarkers for Diabetic Retinopathy using Sequence Mining Techniques

Bioinformatics and sequence mining are the application and development of data mining techniques to solve problems by comprehending biological data. Sequence analysis is the most primitive operation in sequence mining techniques. Modern sequence mining research is specialized in analyzing sequential patterns which are relevant and distinct from one another and utilizing retrieved sequences similarity and distance between different protein sequences can be analyzed. Diabetic retinopathy is the major cause of blindness in individuals mostly adults with diabetes and is it is the common problem of diabetes mellitus across the world. Various research analyses stated that there are many proteins which are found to take part in diabetic retinopathy. In this paper, we have evaluated certain proteins which are closely related with diabetic retinopathy with the help of multiple alignment tool viz. Clustal Omega and obtained a phylogenetic tree of 28 protein sequences gathered from National Center for Biotechnology Information (NCBI). In this work data mining technique called sequence mining plays a significant role in providing phylogram obtained with Neighbor-Joining algorithm. From the phylogenetic tree it was recognized that cortistatin, vitamin-D receptor and somatostatin proteins has close connection with diabetic retinopathy. Molecular docking studies have also been performed which is the most extensively used method for the calculation of protein-ligand interactions. In silico docking studies indicated that four inhibitory compounds i.e. Quercetin, Kaempferol, Naringenin and Melicitrin interact with aldose reductase which also found to have role in diabetic retinopathy. Outcomes infer that techniques intended to standardize cortistatin, vitamin-D receptor and somatostatin activities be of huge advantage and provide benefit in inhibiting diabetic retinopathy.

Medicinal Materials DNA Barcode Database (MMDBD) version 1.5-one-stop solution for storage, BLAST, alignment and primer design.

Authentication of medicinal materials by deoxyribonucleic acid (DNA) technology is gaining popularity. In 2010, our team has created Medicinal Materials DNA Barcode Database (MMDBD) version 1.0 to provide an interactive database for documenting DNA barcode sequences of medicinal materials. This database now contains DNA barcode sequences of medicinal materials listed in the Chinese Pharmacopoeia, Dietary Supplements Compendium and Herbal Medicine Compendium of the US Pharmacopoeia and selected adulterants. The data archive is regularly updated and currently it stores 62 011 DNA sequences of 2111 medicinal materials. Our team has recently completed the major improvement on the interfaces and incorporated essential bioinformatics tools to facilitate the authentication work. MMDBD version 1.5 contains detailed information of each medicinal material including their material names, medical part, pharmacopeia information, biological classification in rank of family and status on the Convention on International Trade in Endangered Species of Wild Fauna and Flora and the International Union for Conservation of Nature’s Red List of Threatened Species, if any. DNA sequences can be retrieved by search in Latin scientific name, Chinese name, family name, material name, medical part and simplified Chinese character stroke. A `BLAST’-based engine for searching DNA sequences is included in the MMDBD version 1.5. Since primer design is a key step in DNA barcoding authentication, we have integrated the `Clustal Omega alignment tool’ and `Primer3’ in the form of web interface. These new tools facilitate multiple sequence comparison and the design of primers for amplification of a target DNA barcode region, allowing DNA barcoding authentication.

UTILIZATION OF INTERNAL TRANSCRIBED SPACER (ITS) AS A MOLECULAR MARKER FOR PHYLOGENETIC RELATIONSHIP OF Solanaceae FAMILY

Solanaceae family is considered one of the major plant families providing food. Studies based on DNA and RNA molecular markers are more precise, reliable and powerful to assess the relationships between species also between genera in the same family. ITS sequences have been broadly used in the inference of phylogenetic hypotheses and in molecular evolution studies of plants, because it is variable, represent point mutations and insertions/deletions (indels). Specific ITS fragments were produced using the universal primer through seven sample of Solanaceae. Purified fragments were sequenced and each sequence was aligned individually at BLAST to confirm each species and to determine its relation to other sequence. Multiple Sequence alignment was done using Clustal Omega program for the seven sequences, and phylogenetic relationship showed that Potato was closely related to Tomato as they were grouped in one main cluster. Whereas Eggplant was related to both type of pepper (Chili and Bell) and was grouped in another main cluster. The Ground Cherry was separated alone in the third main cluster. Finally, ten related sequences picked from each alignment were deduced from BLAST to make the multiple alignments for the seven studied samples with other species of Solanaceae family.

Using DNA Barcodes to Evaluate Ecosystem Health in the SWRCMS Reserve

The Western Center Academy (WCA) Charter School and Mt. San Jacinto College (MSJC) are located on the Southwestern Riverside County Multi-Species Reserve (SWRCMSR). Developed in 1992, the reserve is home to at least 8 sensitive bird, animal, and plant species. Although the reserve is protected against direct human influence, within a 100-mile circle of SWRCMSR live more than 21 million people with their indirect impact on the environment. Our goal was to determine if the reserve is protecting the species within by analyzing the genetics of those species to determine if there is a healthy amount of genetic diversity in the populations within. We collected insects at MSJC Menifee campus and in the SWRCMSR to evaluate how human impact affects the genetic diversity of insects. MSJC served as the site that was impacted by human activities and SWRCMSR served as the site that was not impacted by human activities. The School Malaise Trap Project (SMTP) selected 160 of our specimens, sequenced their genetic barcodes, and entered the data into the Barcode of Life Database (BOLD) for analysis. Using the BOLD website and a Clustal Omega tool, we compared the genetic sequences in the insects we collected to measure genetic differences in the two populations of insects. The results from the two collections seem to show that the nature reserve insect population is a younger population with genetic changes possibly acquired by gene flow from immigration. The insect population at MSJC appears to be well developed, but isolated genetically.

Exploration of genetic polymorphism in targeted region of Protein Phosphatase 1 Regulatory subunit 11 (PPP1R11) in Murrah bulls

The present study was carried out with the objective to explore genetic polymorphism in Protein Phosphatase 1 Regulatory subunit 11 (PPP1R11) gene for its possible association with conception rate in Murrah bulls. Genomic DNA was isolated by phenol-chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-NDRI, Karnal. The quality and concentration of DNA was checked through spectrophotometer reading. The target region of PPP1R11 gene was amplified using 3 sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced, edited and aligned using Clustal Omega with Bubalus bubalis for identification of SNPs. A total of 7 SNPs were found, out of which 1 at exon 3 and 6 at 3’UTR. SNP in exon 3 at nt.G1932C resulted in non synonymous (Serine to Threonine) type of amino acid substitution at position 109 of PPP1R11 gene in Murrah. PCR-RFLP was carried out using restriction enzyme BsmAI for genotyping the animals. All Murrah animals included in present study were monomorphic with CC genotype for the targeted region of PPP1R11 gene.

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise.

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Clustal Omega for making accurate alignments of many protein sequences.

Clustal Omega is a widely used package for carrying out multiple sequence alignment. Here, we describe some recent additions to the package and benchmark some alternative ways of making alignments. These benchmarks are based on protein structure comparisons or predictions and include a recently described method based on secondary structure prediction. In general, Clustal Omega is fast enough to make very large alignments and the accuracy of protein alignments is high when compared to alternative packages. The package is freely available as executables or source code from www.clustal.org or can be run on-line from a variety of sites, especially the EBI www.ebi.ac.uk.

Acetylation and methylation of sperm histone 3 lysine 27 (H3K27ac and H3K27me3) are associated with bull fertility

Epigenetic modifications in histones are crucial for proper sperm physiology, egg activation and reproductive development of males. The objectives of this study were to determine the conservation and interactomes of histone three (H3) and ascertain the expression dynamics of acetylated and methylated H3 lysine 27 (H3K27ac and H3K27me3) in spermatozoa from Holstein bulls with different fertility. Methods in immunocytochemistry and flow cytometry were used to evaluate the expression dynamics of H3K27ac and H3K27me3 in spermatozoa from 10 bulls with different in vivo fertility. Computational biology methods including Clustal Omega and Cytoscape were performed to determine the evolutionary conservation and interactome of H3. The post-translational modifications (PTM) of H3 (H3K27ac and H3K27me3) had different spatiotemporal dynamics in the sperm head. Intensities of methylation were higher than those of acetylation and inversely correlated between the two fertility groups (p=.0032). The interacting proteins of H3 are involved in critical subcellular processes such as regulation of methylation, nucleosome assembly, regulation of DNA replication and chromatin assembly. These results are significant because they help advance fundamental science and biotechnology of mammalian reproduction.

Applying MSSIM combined chaos game representation to genome sequences analysis

Converting DNA sequence to image by using chaos game representation (CGR) is an effective genome sequence pretreatment technology, which provides the basis for further analysis between the different genes. In this paper, we have constructed 10 mammal species, 48 hepatitis E virus (HEV), and 10 kinds of bacteria genetic CGR images, respectively, to calculate the mean structural similarity (MSSIM) coefficient between every two CGR images. From our analysis, the MSSIM coefficient of gene CGR images can accurately reflect the similarity degrees between different genomes. Hierarchical clustering analysis was used to calculate the class affiliation and construct a dendrogram. Large numbers of experiments showed that this method gives comparable results to the traditional Clustal X phylogenetic tree construction method, and is significantly faster in the clustering analysis process. Meanwhile MSSIM combined CGR method was also able to efficiently clustering of large genome sequences, which the traditional multiple sequence alignment methods (e.g. Clustal X, Clustal Omega, Clustal W, et al.) cannot classify.

P071 The reference: Sequence-level comparison of HLA loci in unrelated hematopietic cell transplant (HCT) pairs

Aim HLA matching by allele-level genotyping is largely based on genetic similarity between exons which encode the antigen recognition site (ARS). Next-generation sequencing can identify polymorphisms in non-ARS exons, introns, and untranslated regions, but the impact on HCT outcome is unclear. Here we report a bioinformatics approach to directly compare recipient and donor HLA sequences and identify regions of genetic dissimilarity. Methods The Illumina TruSight HLA NGS assay was used to sequence the HLA-A, -B, -C, DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 loci for 169 paired donor and recipient samples obtained from the CIBMTR repository. Assign v2.1 software was used to generate HLA genotypes and consensus sequences from each locus. Recipient consensus sequences are aligned using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo ) to both the corresponding reference allele in IMGT/HLA v3.27.0 and the donor consensus sequence for that locus. Mismatches between consensus sequences are annotated according to genetic features (exon, intron, UTR) obtained from the IMGT/HLA reference allele. Indels are considered when adjusting genetic feature locations. Exonic sequence alignments and amino acid substitutions are then confirmed by comparison with IMGT/HLA. Alignments are visualized using custom software to highlight nucleotide and amino acid differences and provide summary statistics. Results The algorithm was validated using HCT pairs with known allele-level mismatches to confirm correct assignment of ARS differences. The algorithm accurately identifies and categorizes HCT pairs with HLA loci that differ outside the ARS region as well as those that do not. In addition, the algorithm discovers instances where the consensus phase genotype is ambiguous. Conclusions We report a novel bioinformatics approach to directly compare donor and recipient consensus HLA sequences to identify and visualize regions of sequence heterogeneity. Furthermore, our automated pipeline aids in the discovery of novel “feature-specific” mismatch patterns, offering an additional means of comparison beyond traditional HLA allele assignment. We plan to utilize this approach to categorize HCT pairs for analysis of clinical outcome measures by matching status at each region of interest.

Behçet Disease serum is immunoreactive to neurofilament medium which share common epitopes to bacterial HSP-65, a putative trigger

Autoimmune and dysimmune inflammatory mechanisms on a genetically susceptible background are implicated in the etiology of Behçet's Disease (BD). Heat-shock protein-65 (HSP-65) derived from Streptococcus sanguinis was proposed as a triggering factor based on its homology with human HSP-60. However, none of the autoantigens identified so far in sera from BD share common epitopes with bacterial HSP-65 or has a high prevalence. Here, we report that sera from BD patients are immunoreactive against filamentous neuronal processes in the mouse brain, retina and scrotal skin in great majority of patients. By using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting, Western blotting and peptide blocking experiments, we have identified neurofilament medium (NF-M) as the probable antigen for the serologic response observed. Clustal Omega analyses detected significant structural homology between the human NF-M and bacterial HSP-65 corresponding to amino acids 111–126, 213–232 and 304–363 of mycobacterial HSP-65, which were previously identified to induce proliferation of lymphocytes obtained from BD patients. We also found that sera immunoreactive against NF-M cross-reacted with bacterial HSP-65. These findings suggest that NF-M may be involved in autoimmunity in BD due to its molecular mimicry with bacterial HSP-65.

Bacterial Foraging Optimization \u2013Genetic Algorithm for Multiple Sequence Alignment with Multi-Objectives

This research work focus on the multiple sequence alignment, as developing an exact multiple sequence alignment for different protein sequences is a difficult computational task. In this research, a hybrid algorithm named Bacterial Foraging Optimization-Genetic Algorithm (BFO-GA) algorithm is aimed to improve the multi-objectives and carrying out measures of multiple sequence alignment. The proposed algorithm employs multi-objectives such as variable gap penalty minimization, maximization of similarity and non-gap percentage. The proposed BFO-GA algorithm is measured with various MSA methods such as T-Coffee, Clustal Omega, Muscle, K-Align, MAFFT, GA, ACO, ABC and PSO. The experiments were taken on four benchmark datasets such as BAliBASE 3.0, Prefab 4.0, SABmark 1.65 and Oxbench 1.3 databases and the outcomes prove that the proposed BFO-GA algorithm obtains better statistical significance results as compared with the other well-known methods. This research study also evaluates the practicability of the alignments of BFO-GA by applying the optimal sequence to predict the phylogenetic tree by using ClustalW2 Phylogeny tool and compare with the existing algorithms by using the Robinson-Foulds (RF) distance performance metric. Lastly, the statistical implication of the proposed algorithm is computed by using the Wilcoxon Matched-Pair Signed- Rank test and also it infers better results.

Phylogenetic Analysis of RuBIsCO: An Active Learning Strategy for Teaching Plant Evolution

Lane College is a small historically black college located in Jackson, TN. The majority of the students are underserved African Americans from areas of high poverty. General Biology at Lane College is a foundational survey course required for Biology majors and Physical Education majors. Topics covered in the second semester of the course include natural selection, evolution, biodiversity, and ecology. This course is especially challenging for students given the large amount of information that they are typically required to memorize. We are currently implementing a peer‐led team learning strategy to enable students to actively engage in learning core concepts outlined in Vision and Change. The learning goal for the exercise presented here is to explore the evolutionary relatedness of different plant species. The learning objectives for this exercise are for students to understand plant taxonomy and to analyze the DNA evidence for the origin of the chloroplast through a phylogenetic comparison of the ribulose‐1,5 bisphosphate carboxylase/oxidase large subunit (rbcl) gene and to understand plant taxonomy. Prior to coming to class, the students are given a reading assignment for the Land Plants chapter in their textbook. During the first class meeting, at the beginning of the exercise, the students are divided into teams of four. Each team is assigned a set of four unknown DNA sequences from closely related plant or algal species, and each student chooses a sequence. Each student then uses the standard Basic Alignment Search Tool (BLAST) to identify the gene and their plant species for their sequence. The students then answer specific questions about their plant or algal species regarding taxonomic classification. Next, each student creates a sequence alignment of the entire class set of sequences and constructs a phylogenetic tree using CLUSTAL Omega. They then compare their phylogenetic tree with the one from their textbook and discuss the similarities and differences between the trees. At the next class meeting, each group presents a short summary of the characteristics for their assigned plant clade.Support or Funding InformationNational Science Foundation: HRD 1623340

Dissemination of metallo-β-lactamase in Pseudomonas aeruginosa isolates in Egypt: mutation in blaVIM-4.

This study was designed to investigate the prevalence of metallo-β-lactamase (MBL) in Pseudomonas aeruginosa isolates collected from Suez Canal University Hospital in Ismailia, Egypt. Antibiotic susceptibility testing and phenotypic and genotypic screening for MBLs were performed on 147 isolates of P. aeruginosa. MICs were determined by agar dilution method for carbapenem that was ≥2 μg/mL for meropenem. MBL genes were detected by multiplex and monoplex PCR for P. aeruginosa-harbored plasmids. Mutation profile of sequenced MBL genes was screened using online software Clustal Omega. Out of 147 P. aeruginosa, 39 (26.5%) were carbapenem-resistant isolates and 25 (64%) were confirmed to be positive for MBLs. The susceptibility rate of P. aeruginosa toward polymyxin B and norfloxacin was 99% and 88%, respectively. Identification of collected isolates by API analysis and constructed phylogenetic tree of 16S rRNA showed that the isolates were related to P. aeruginosa species. The frequency of blaGIM-1, blaSIM-1, and blaSPM-1 was 52%, 48%, and 24%, respectively. BlaVIM and blaIMP-like genes were 20% and 4% and the sequences confirm the isolate to be blaVIM-1, blaVIM-2, blaVIM-4, and blaIMP-1. Three mutations were identified in blaVIM-4 gene. Our study emphasizes the high occurrence of multidrug-resistant P. aeruginosa-producing MBL enzymes.

Clustering of fungal hexosaminidase enzymes based on free alignment method using MLP neural network

Studies of biological evolution have generally focused on nucleotide or amino acid sequences of certain genes related to specific enzymes. Most phylogenetic tree constructions have been carried out using amino acid sequences and are used as a predictor to show evolutionary relationships. Phylogenetic analysis is usually performed based on multiple sequence alignment of a gene from different organisms including fungi. A number of programs have been introduced for gene clustering and phylogenetic analysis. For example, the most popular web-based program is Clustal Omega which is commonly used by biologists. When the number of uploaded sequences increases, this program not only works slowly but also the final constructed cladogram is confusing and incorrect from evolutionary point of view. In the present study, we used fungal hexosaminidases which are extracellular enzymes with a lot of applications in biotechnology but extremely varied and confusing in evolutionary terms. A standard taxonomy-based phylogenetic tree was constructed for 835 FH amino acid sequences retrieved from National Center for Biotechnology Information (NCBI) on March 16, 2015. Then a supervised multilayer perceptron (MLP) neural network was used to discriminate FH sequences. Based on relative frequency of amino acid in FH sequences, 41 neural networks were designed for seven levels from the phylum to family. Minimum accuracy of the neural network was equal to 99% at all seven discrimination levels. As a final step, an additional evaluation was performed on the designed model with 143 new released FH sequences extracted on July 1, 2015. The clustering results have shown a proper match with fungal taxonomy to show evolutionary relationships.

Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls.

Aim:This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls.Materials and Methods:Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme.Results:The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study.Conclusion:A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible.