Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) fromAegilops tauschii

Genome ◽  
2006 ◽  
Vol 49 (7) ◽  
pp. 735-745 ◽  
Author(s):  
Yanzhen Zhang ◽  
Qiaoyun Li ◽  
Yueming Yan ◽  
Jigang Zheng ◽  
Xueli An ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Aegilops Tauschii ◽  
Pcr Primers ◽  
Duplication Event ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS–PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF–MS). The gene encoding the HMM subunit Dy10.1twas amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1tsubunit gene consisted of 1980 bp encoding a protein of 658 residues with an Mrsof 68 611 Da, which was similar to the Mrsdetermined by MALDI–TOF–MS. The deduced amino acid sequence indicated that Dy10.1tsubunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1tgene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.Key words: HMW glutenin genes, MALDI-TOF-MS, AS-PCR, cSNP, phylogenetic analysis, Aegilops tauschii.

scholarly journals Detection of Structural and Conformational Changes in ALS-Causing Mutant Profilin1 With Hydrogen/Deuterium Exchange Mass Spectrometry and Bioinformatics Techniques

2021 ◽  
Author(s):  
Ahmad Shahir Sadr ◽  
zahra abdollahpour ◽  
Atousa Aliahmadi ◽  
Changiz Eslahchi ◽  
Mina Nekouei ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Amino Acid ◽  
Conformational Changes ◽  
Docking Simulation ◽  
Hydrogen Deuterium Exchange ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Hydrogen Deuterium ◽  
Tof Ms

Abstract The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution in the structural changes of profilin1 (PFN1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to Valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN1 protein dynamic behavior using purified wild type and mutant PFN1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN1WT and PFN1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN1s’ dimerization, oligomerization, and aggregation.

scholarly journals Large-scale PRNP genotyping of small ruminants using an automated high-throughput MALDI-TOF MS assay

2019 ◽  
Vol 31 (4) ◽  
pp. 629-633
Author(s):  
Sergio Migliore ◽  
Maurizio Bivona ◽  
Enrico Gagliostro ◽  
Onofrio Buttitta ◽  
Francesca Lo Mascolo ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Genetic Resistance ◽  
Small Ruminants ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Automated Processing ◽  
Tof Ms

Scrapie resistance or susceptibility in sheep is associated with single nucleotide polymorphisms (SNPs) at codons 136, 154, and 171 of the prion protein gene ( PRNP). In addition, phenylalanine mutation at codon 141 has been recognized as a risk factor for atypical scrapie. In contrast, K222, D146, and S146 alleles confer genetic resistance to classical scrapie in goats. High-throughput genotyping technologies would provide significant benefits in scrapie eradication plans. The ability to resolve oligonucleotides varying in mass by less than a single nucleotide makes MALDI-TOF mass spectrometry (MS) a suitable platform for PRNP genotyping. We evaluated the commercial Myriapod scrapie kit (Diatech Pharmacogenetics), associated with a highly automated processing platform incorporating MALDI-TOF MS technology, to detect SNPs at codons 136, 154, 171, 141, and 222 of small ruminant PRNP. The Myriapod scrapie kit was accredited according to UNI CEI EN ISO/IEC 17025. We present the genotyping results of 10,960 sheep in Sicily and 1,822 goats in Sicily and Calabria (southern Italy) tested during 2017. We found a high frequency (43.9%) of the protective ARR allele in sheep and a promising 12.3% of the resistant K222 variant in goats. This efficient and high-throughput method is suitable for extensive PRNP genotyping, as requested in the European scrapie eradication plan.

scholarly journals Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers

2010 ◽  
Vol 76 (18) ◽  
pp. 6248-6256 ◽  
Author(s):  
Annette Wensing ◽  
Stefan Zimmermann ◽  
Klaus Geider
Keyword(s):  
Mass Spectrometry ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
Cell Protein ◽  
Whole Cell ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Pantoea Stewartii ◽  
Ms Analysis ◽  
Tof Ms

ABSTRACT Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA.

scholarly journals MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms

2007 ◽  
Vol 53 (1) ◽  
pp. 24-33 ◽  
Author(s):  
Julia K Blievernicht ◽  
Elke Schaeffeler ◽  
Kathrin Klein ◽  
Michel Eichelbaum ◽  
Matthias Schwab ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphisms ◽  
Direct Sequencing ◽  
Plasma Concentrations ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Ms ◽  
Hepatic Expression ◽  
Maldi Tof ◽  
Tof Ms

Abstract Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. Results: Five multiplex assays were developed, comprising the following 15 SNPs: −82T→C (*22); 86G→C (R29T, *17); 136A→G (M46V, *11); 296G→A (G99E, *12); 415A→G (K139E, *8, *13); 419G→A (R140Q, *14); 516G→T (Q172H, *6, *7, *9, *13, *19, *20), 547G→A (V183I); 769G→A (D257N); 785A→G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T→C (I328T, *16, *18); 1006C→T (R336C, *19); 1172T→A (I391N, *15); 1282C→A (P428T, *21); 1459C→T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. Conclusions: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.

scholarly journals Evaluation of MALDI–TOF Mass Spectrometry in Diagnostic and Environmental Surveillance of Legionella Species: A Comparison With Culture and Mip-Gene Sequencing Technique

2020 ◽  
Vol 11 ◽  
Author(s):  
Maria Rosaria Pascale ◽  
Marta Mazzotta ◽  
Silvano Salaris ◽  
Luna Girolamini ◽  
Antonella Grottola ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Phylogenetic Analysis ◽  
Legionella Pneumophila ◽  
Gene Sequencing ◽  
Species Level ◽  
Environmental Surveillance ◽  
Routine Diagnostics ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Legionella spp. are widespread bacteria in aquatic environments with a growing impact on human health. Between the 61 species, Legionella pneumophila is the most prevalent in human diseases; on the contrary, Legionella non-pneumophila species are less detected in clinical diagnosis or during environmental surveillance due to their slow growth in culture and the absence of specific and rapid diagnostic/analytical tools. Reliable and rapid isolate identification is essential to estimate the source of infection, to undertake containment measures, and to determine clinical treatment. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS), since its introduction into the routine diagnostics of laboratories, represents a widely accepted method for the identification of different bacteria species, described in a few studies on the Legionella clinical and environmental surveillance. The focus of this study was the improvement of MALDI–TOF MS on Legionella non-pneumophila species collected during Legionella nosocomial and community surveillance. Comparative analysis with cultural and mip-gene sequencing results was performed. Moreover, a phylogenetic analysis was carried out to estimate the correlations amongst isolates. MALDI–TOF MS achieved correct species-level identification for 45.0% of the isolates belonging to the Legionella anisa, Legionella rubrilucens, Legionella feeleii, and Legionella jordanis species, displaying a high concordance with the mip-gene sequencing results. In contrast, less reliable identification was found for the remaining 55.0% of the isolates, corresponding to the samples belonging to species not yet included in the database. The phylogenetic analysis showed relevant differences inside the species, regruped in three main clades; among the Legionella anisa clade, a subclade with a divergence of 3.3% from the main clade was observed. Moreover, one isolate, identified as Legionella quinlivanii, displayed a divergence of 3.8% from the corresponding reference strain. However, these findings require supplementary investigation. The results encourage the implementation of MALDI–TOF MS in routine diagnostics and environmental Legionella surveillance, as it displays a reliable and faster identification at the species level, as well as the potential to identify species that are not yet included in the database. Moreover, phylogenetic analysis is a relevant approach to correlate the isolates and to track their spread, especially in unconventional reservoirs, where Legionella prevention is still underestimated.

scholarly journals Isolation and amino acid sequencing by MALDI-TOF-MS/MS of a novel antimicrobial anionic peptide from the skin secretion of Osteocephalus taurinus (Anura, Hylidae)

2012 ◽  
Vol 23 (12) ◽  
pp. 2133-2136 ◽  
Author(s):  
Túlio O. G. Costa ◽  
Richardson A. Almeida ◽  
Jorge T. Melo ◽  
Hector H. F. Koolen ◽  
Felipe M. A. da Silva ◽  
...  
Keyword(s):  
Amino Acid ◽  
Skin Secretion ◽  
Amino Acid Sequencing ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

scholarly journals Emergence of multi-drug resistance Candida auris in Saudi Arabia

2020 ◽  
Author(s):  
Reem AlJindan ◽  
Doaa Mostafa AlEraky ◽  
Nehal Mahmoud ◽  
Sayed AbdulAzeez ◽  
J.Francis Borgio
Keyword(s):  
Phylogenetic Analysis ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Multi Drug Resistance ◽  
Rrna Gene ◽  
Candida Auris ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Resistant Strains ◽  
Tof Ms

Abstract Background: Candida auris is an emerging multi-drug resistant pathogen with high mortality rate, several cases and nosocomial infections have been reported worldwide causing a major challenge for clinicians and microbiological laboratories. Objectives: The study aim to, describe new cases of C. auris from different sites of infection confirmed by MALDI TOF MS and ribosomal sequencing, develop the phylogenetic analysis of these isolates, detect resistant strains of C. auris to both azole and echinocandin by amplification of ERG11 and FKS1 genes. Methods: A total of six specimens were collected from blood, urine, ear swab and groin screening sample during the period from November 2018 to March 2019. Isolates were incubated for 48 hours on Sabroud agar at 42℃, then confirmed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS). Sequences of 18S rRNA gene from isolates and phylogenetic analysis were performed. Finally, molecular analysis of resistance genes was performed to detect the efficacy of treatment. Results: Clinical isolates were identified by the growth on Sabroud agar at 42℃ and confirmed by MALDI-TOF MS analysis. Evolutionary analyses were conducted in MEGA7 and sequences of 18S rRNA gene were submitted to GenBank. All samples were positive for both ERG11 and FKS1 which confer azole- and echinocandin-resistant strains. Conclusions: This study shed light on a public health threat of an emerging pathogen. Therefore, the hospital implemented strict contact screening and infection control precautions to prevent C. auris infection. Finally, there is a critical need to monitor the antifungal resistance in different geographical areas and implementation of efficient guidelines for treatment.

Characterization and comparative analysis of three low molecular weight glutenin C-subunit genes isolated from Aegilops tauschii

2007 ◽  
Vol 87 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Yu-He Pei ◽  
Ai-Li Wang ◽  
Xue-Li An ◽  
Xiao-Hui Li ◽  
Yan-Zhen Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Aegilops Tauschii ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Low Molecular Weight ◽  
Primitive Form ◽  
Reading Frame ◽  
Specific Pcr

Three low molecular weight glutenin subunit (LMW-GS) genes from T121, T128 and T132 accessions of Aegilops tauschii (DD, 2n = 2x = 14) were amplified using allelic-specific PCR primers. The amplified products with a size of about 900 bp were cloned and sequenced. Three complete coding sequences of LMW-GS with 918 bp, 921 bp and 918 bp were obtained and named as LMW-T121, LMW-T128, LMW-T132, respectively. Each gene contained a complete open reading frame and had no introns. The deduced amino acid sequences showed that all belonged to LMW-m type subunit with a predicted molecular weight of about 32 kDa, corresponding to the size of LMW C-subunits. All three subunits possessed eight cysteine residues and had greater homology with previously characterized LMW-m subunits from bread wheat and related species than LMW-s or LMW-i sequences. Some amino acid substitutions and insertion/deletion variations among the sequences were detected. The corresponding three C-subunits in seed endosperm encoded by LMW-T121, LMW-T128, LMW-T132, respectively, were identified and confirmed by SDS-PAGE, MALDI-TOF-MS and direct N-terminal amino acid sequencing. Phylogenetic analysis demonstrated that LMW-m and LMW-s type subunit genes possessed higher identity and they were obviously separated from LMW-i type subunit genes. The LMW-m type might be the primitive form while the LMW-s and LMW-i types are variant forms. Key words: Aegilops tauschii, LMW-GS, AS-PCR, phylogenetic analysis

Sign in / Sign up

Export Citation Format

Share Document