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https://www.readbyqxmd.com/read/29329457/a-novel-application-of-rnase-h2-dependent-quantitative-pcr-for-detection-and-quantification-of-grosmannia-clavigera-a-mountain-pine-beetle-fungal-symbiont-in-environmental-samples
#1
Chandra H McAllister, Colleen E Fortier, Kate R St Onge, Bianca M Sacchi, Meaghan J Nawrot, Troy Locke, Janice E K Cooke
Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry)...
January 10, 2018: Tree Physiology
https://www.readbyqxmd.com/read/29322447/characterization-of-circular-rna-concatemers
#2
Thomas B Hansen
Circular RNAs (circRNAs) constitute a novel subset in the fascinating world of noncoding RNA, and they are found in practically all eukaryotes. Most of them exhibit low expression levels but some are extremely abundant. Typically, circRNAs are studied by RT-PCR-based assays, but for certain types of analyses this technique is not suitable. Circular RNA with repetitive exons (circular concatemers) has been observed by us and others when transiently expressing circRNAs in cells, however techniques and assays to study these species have not been established...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29322445/northern-blot-analysis-of-circular-rnas
#3
Tim Schneider, Silke Schreiner, Christian Preußer, Albrecht Bindereif, Oliver Rossbach
Northern blotting enables the specific detection and characterization of RNA molecules. Recently, circular RNAs (circRNAs) were described as a new class of cell type-specific noncoding RNAs. With the discovery of many novel circRNAs on the basis of high-throughput sequencing and bioinformatics, a solid biochemical approach is required to directly detect and validate specific circRNA species. Here we give a detailed overview of how different Northern blot methods can be employed to validate specific circRNAs...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29306413/relative-quantitation-of-neutral-and-sialylated-n-glycans-using-stable-isotopic-labeled-d0-d5-benzoyl-chloride-by-maldi-ms
#4
Chang Wang, Yike Wu, Liang Zhang, Bi-Feng Liu, Yawei Lin, Xin Liu
Quantitative analysis of glycans is an emerging field in glycomic research. Herein we present a rapid and effective dual-labeling strategy, in the combination of isotopic derivatization of N-glycosylamine-based glycans by d0/d5-benzoyl chloride and methylamidation of sialic acids, to relatively quantify both neutral and sialylated N-glycans simultaneously by MALDI-MS. The derivatization efficiencies were increased by microwave-accelerated deglycosylation which not only largely reduce the time of glycoprotein deglycosylation but also inhibit the hydrolysis of intermediate glycosylamines produced by PNGase F digestion...
March 9, 2018: Analytica Chimica Acta
https://www.readbyqxmd.com/read/29295939/structure-of-hiv-1-reverse-transcriptase-cleaving-rna-in-an-rna-dna-hybrid
#5
Lan Tian, Min-Sung Kim, Hongzhi Li, Jimin Wang, Wei Yang
HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å...
January 2, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29295617/mno2-nanosheet-powered-janus-protection-dna-nanomachines-supporting-robust-rna-imaging
#6
Feng Chen, Min Bai, Yue Zhao, Ke Cao, Xiaowen Cao, Yongxi Zhao
Both biomarker and probe degradations cause serious false assay results. Yet, protecting target or target/probe simultane-ously has rarely been explored. Herein, MnO2 nanosheet-powered target/probe Janus protection DNA nanomachines are reported. It is formed by RNA-responsive assembly of chemically modified two DNA partzymes and one substrate probe in living cells. MnO2 nanosheets are used to facilitate cellular uptake of DNA reagents and generate Mn2+ as indispensable DNAzyme cofactors for efficient catalytic cleavage...
January 3, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29281624/the-smc5-6-complex-regulates-the-yeast-mph1-helicase-at-rna-dna-hybrid-mediated-dna-damage
#7
Juan Lafuente-Barquero, Sarah Luke-Glaser, Marco Graf, Sonia Silva, Belén Gómez-González, Arianna Lockhart, Michael Lisby, Andrés Aguilera, Brian Luke
RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci...
December 27, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/29258713/1-5-disubstituted-1-2-3-triazole-linked-disaccharides-metal-free-syntheses-and-screening-of-a-new-class-of-ribonuclease-a-inhibitors
#8
Anirban Kayet, Dhrubajyoti Datta, Ashrukana Das, Swagata Dasgupta, Tanmaya Pathak
1,5-Regioisomeric triazole linked disaccharides have been synthesized and screened for their inhibitory properties against ribonuclease A (RNase A). The angular constraint-driven 'crescent shaped' inhibitors accommodated themselves into the enzyme active site. An improved enzyme inhibition was observed with increased H-bonding ability of polar functional groups in the modified disaccharides. In this series, introduction of two carboxyl groups in the furanose rings elicited the best result with an inhibition constant of 50 ± 3 µM...
December 5, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/29237844/hsv-1-dna-polymerase-rnase-h-activity-acts-in-a-3-5-direction-and-is-dependent-on-the-3-5-exonuclease-active-site
#9
Jessica L Lawler, Purba Mukherjee, Donald M Coen
The catalytic subunit (Pol) of herpes simplex virus-1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA:DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3' -5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3' -5' exonuclease-deficient mutant, D368A, for DNA polymerase activity, 3' -5' exonuclease activity, and RNase H activity in vitro Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini...
December 13, 2017: Journal of Virology
https://www.readbyqxmd.com/read/29181860/detection-of-helicobacter-pylori-in-stool-samples-of-young-children-using-real-time-polymerase-chain-reaction
#10
Gany Beer-Davidson, Musa Hindiyeh, Khitam Muhsen
BACKGROUND: The aims of this study were to develop and validate a multiplex real-time polymerase chain reaction (q-PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori-positive samples. MATERIALS AND METHODS: Archived stool samples from 188 children aged 6-9 years and 272 samples of 92 infants aged 2-18 months were tested for H. pylori antigens using enzyme immunoassay (EIA)...
November 27, 2017: Helicobacter
https://www.readbyqxmd.com/read/29179918/when-dna-topology-turns-deadly-rna-polymerases-dig-in-their-r-loops-to-stand-their-ground-new-positive-and-negative-super-twists-in-the-replication-transcription-conflict
#11
REVIEW
Andrei Kuzminov
Head-on replication-transcription conflict is especially bitter in bacterial chromosomes, explaining why actively transcribed genes are always co-oriented with replication. The mechanism of this conflict remains unclear, besides the anticipated accumulation of positive supercoils between head-on-conflicting polymerases. Unexpectedly, experiments in bacterial and human cells reveal that head-on replication-transcription conflict induces R-loops, indicating hypernegative supercoiling [(-)sc] in the region - precisely the opposite of that assumed...
November 24, 2017: Trends in Genetics: TIG
https://www.readbyqxmd.com/read/29177738/detection-of-oric-independent-replication-in-escherichia-coli-cells
#12
Makisha Martel, Aurélien Balleydier, Julien Brochu, Marc Drolet
In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29169831/next-generation-sequencing-elucidates-cacao-badnavirus-diversity-and-reveals-the-existence-of-more-than-ten-viral-species
#13
E Muller, S Ravel, C Agret, F Abrokwah, H Dzahini-Obiatey, I Galyuon, K Kouakou, E C Jeyaseelan, J Allainguillaume, A Wetten
Cacao swollen shoot virus is a member of the family Caulimoviridae, genus Badnavirus and is naturally transmitted to Theobroma cacao (L.) by several mealybug species. CSSV populations in West African countries are highly variable and genetically structured into several different groups based on the diversity in the first part of ORF3 which encodes the movement protein. To unravel the extent of isolate diversity and address the problems of low titer and mixed viral sequences in samples, we used Illumina MiSeq and HiSeq technology...
November 21, 2017: Virus Research
https://www.readbyqxmd.com/read/29163435/evolutionary-analysis-of-hiv-1-pol-proteins-reveals-representative-residues-for-viral-subtype-differentiation
#14
Shohei Nagata, Junnosuke Imai, Gakuto Makino, Masaru Tomita, Akio Kanai
RNA viruses have been used as model systems to understand the patterns and processes of molecular evolution because they have high mutation rates and are genetically diverse. Human immunodeficiency virus 1 (HIV-1), the etiological agent of acquired immune deficiency syndrome, is highly genetically diverse, and is classified into several groups and subtypes. However, it has been difficult to use its diverse sequences to establish the overall phylogenetic relationships of different strains or the trends in sequence conservation with the construction of phylogenetic trees...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29152711/the-burst-phase-folding-intermediate-of-ribonuclease-h-changes-conformation-over-evolutionary-history
#15
Shion A Lim, Susan Marqusee
The amino acid sequence encodes the energy landscape of a protein. Therefore, we expect evolutionary mutations to change features of the protein energy landscape, including the conformations adopted by a polypeptide as it folds to its native state. Ribonucleases H (RNase H) from Escherichia coli and Thermus thermophilus both fold via a partially folded intermediate in which the core region of the protein (helices A-D and strands 4-5) is structured. Strand 1, however, uniquely contributes to the T. thermophilus RNase H folding intermediate (Icore+1 ), but not the E...
November 20, 2017: Biopolymers
https://www.readbyqxmd.com/read/29126318/rnase-h-sequence-preferences-influence-antisense-oligonucleotide-efficiency
#16
Lukasz J Kielpinski, Peter H Hagedorn, Morten Lindow, Jeppe Vinther
RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides...
November 8, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29122886/mechanism-of-polypurine-tract-primer-generation-by-hiv-1-reverse-transcriptase
#17
Małgorzata Figiel, Miroslav Krepl, Sangwoo Park, Jarosław Poznański, Krzysztof Skowronek, Agnieszka Gołąb, Taekjip Ha, Jiři Šponer, Marcin Nowotny
HIV-1 reverse transcriptase (HIV-1 RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear...
November 9, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29118447/influenza-virus-segment-5-rna-secondary-structure-and-new-targets-for-antiviral-strategies
#18
Marta Soszynska-Jozwiak, Paula Michalak, Walter N Moss, Ryszard Kierzek, Julita Kesy, Elzbieta Kierzek
Influenza A virus is a threat for humans due to seasonal epidemics and occasional pandemics. This virus can generate new strains that are dangerous through nucleotide/amino acid changes or through segmental recombination of the viral RNA genome. It is important to gain wider knowledge about influenza virus RNA to create new strategies for drugs that will inhibit its spread. Here, we present the experimentally determined secondary structure of the influenza segment 5 (+)RNA. Two RNAs were studied: the full-length segment 5 (+)RNA and a shorter construct containing only the coding region...
November 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29117130/characterization-of-nucleoside-reverse-transcriptase-inhibitor-associated-mutations-in-the-rnase-h-region-of-hiv-1-subtype-c-infected-individuals
#19
Sinaye Ngcapu, Kristof Theys, Pieter Libin, Vincent C Marconi, Henry Sunpath, Thumbi Ndung'u, Michelle L Gordon
The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed...
November 8, 2017: Viruses
https://www.readbyqxmd.com/read/29111294/oxygen-binding-isotope-effects-of-triazole-based-hiv-1-reverse-transcriptase-inhibitors-indicate-the-actual-binding-site
#20
Agnieszka Krzemińska, Tomasz Frączek, P Paneth
Binding isotope effects (BIEs) associated with binding of four triazole-based ligands to HIV-1 reverse transcriptase have been calculated at the QM/MM MD level of theory. Two main binding sites: allosteric cavity and RNase H active site, as well as three other sites reported in the literature (the Knuckles, the NNRTI Adjacent, and Incoming Nucleotide Binding) have been considered. The interactions between inhibitors and these protein sites have been quantified by binding free energies obtained from free energy perturbation (FEP) calculations, supported by interaction energy analysis...
December 1, 2017: Archives of Biochemistry and Biophysics
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