1,199 results on '"Nucleases"'
Search Results
2. Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining.
- Author
-
Stinson, Benjamin M., Carney, Sean M., Walter, Johannes C., and Loparo, Joseph J.
- Subjects
DNA ,NUCLEASES ,ENZYMES - Abstract
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ. Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double strand-break (DSB) repair, directly re-ligates broken DNA ends with minimal errors. In this study, the authors identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) that prioritize ligation and promote NHEJ fidelity. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. Everything OLD is new again: How structural, functional, and bioinformatic advances have redefined a neglected nuclease family.
- Author
-
Dot, Elena Wanvig, Thomason, Lynn C., and Chappie, Joshua S.
- Subjects
- *
NUCLEASES , *FAMILIES , *NEIGHBORHOODS , *ENZYMES , *TAXONOMY - Abstract
Overcoming lysogenization defect (OLD) proteins are a conserved family of ATP‐powered nucleases that function in anti‐phage defense. Recent bioinformatic, genetic, and crystallographic studies have yielded new insights into the structure, function, and evolution of these enzymes. Here we review these developments and propose a new classification scheme to categorize OLD homologs that relies on gene neighborhoods, biochemical properties, domain organization, and catalytic machinery. This taxonomy reveals important similarities and differences between family members and provides a blueprint to contextualize future in vivo and in vitro findings. We also detail how OLD nucleases are related to PARIS and Septu anti‐phage defense systems and discuss important mechanistic questions that remain unanswered. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
4. ClC-7 drives intraphagosomal chloride accumulation to support hydrolase activity and phagosome resolution.
- Author
-
Jing Ze Wu, Zeziulia, Mariia, Whijin Kwon, Jentsch, Thomas J., Grinstein, Sergio, and Freeman, Spencer A.
- Subjects
- *
GLYCOSIDASES , *HYDROLASES , *CHLORIDES , *NUCLEASES , *CHLORIDE channels , *ENZYMES , *AQUAPORINS - Abstract
Degradative organelles contain enzymes that function optimally at the acidic pH generated by the V-ATPase. The resulting transmembrane H+ gradient also energizes the secondary transport of several solutes, including Cl-. We report that Cl- influx, driven by the 2Cl-/H+ exchanger ClC-7, is necessary for the resolution of phagolysosomes formed by macrophages. Cl- transported via ClC-7 had been proposed to provide the counterions required for electrogenic H+ pumping. However, we found that deletion of ClC-7 had a negligible effect on phagosomal acidification. Instead, luminal Cl- was found to be required for activation of a wide range of phagosomal hydrolases including proteases, nucleases, and glycosidases. These findings argue that the primary role of ClC-7 is the accumulation of (phago)lysosomal Cl- and that the V-ATPases not only optimize the activity of degradative hydrolases by lowering the pH but, importantly, also play an indirect role in their activation by providing the driving force for accumulation of luminal Cl- that stimulates hydrolase activity allosterically. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
5. Researchers at Youjiang Medical University for Nationalities Have Reported New Data on Life Science (Construction of a Fluorescence Sensing Platform Based On Risc and Crispr/ Cas12a for the Assay of Ago2 Enzyme Activity)
- Subjects
Nucleic acids ,Fluorescence ,Nucleases ,Enzymes ,Physical fitness ,Health - Abstract
2023 NOV 18 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- Investigators publish new report on Life Science. According to news originating from [...]
- Published
- 2023
6. Eukaryotic-driven directed evolution of Cas9 nucleases
- Subjects
Nucleases ,Enzymes ,DNA binding proteins ,Physical fitness ,Health - Abstract
2023 OCT 7 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- According to news reporting based on a preprint abstract, our journalists obtained [...]
- Published
- 2023
7. Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1
- Subjects
Medical research ,Medicine, Experimental ,Nucleases ,Enzymes ,Physical fitness ,Health - Abstract
2023 JUL 22 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- According to news reporting based on a preprint abstract, our journalists obtained [...]
- Published
- 2023
8. Max-Planck-Institute for Biochemistry Researcher Details New Studies and Findings in the Area of Enzymes and Coenzymes (UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2)
- Subjects
Boehringer Ingelheim GmbH ,Biochemistry ,Nucleases ,RNA ,Enzymes ,Pharmaceutical industry ,Biological sciences ,Health - Abstract
2024 MAY 21 (NewsRx) -- By a News Reporter-Staff News Editor at Life Science Weekly -- Data detailed on enzymes and coenzymes have been presented. According to news originating from [...]
- Published
- 2024
9. Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.
- Author
-
Vicencio, Jeremy, Sánchez-Bolaños, Carlos, Moreno-Sánchez, Ismael, Brena, David, Vejnar, Charles E., Kukhtar, Dmytro, Ruiz-López, Miguel, Cots-Ponjoan, Mariona, Rubio, Alejandro, Melero, Natalia Rodrigo, Crespo-Cuadrado, Jesús, Carolis, Carlo, Pérez-Pulido, Antonio J., Giráldez, Antonio J., Kleinstiver, Benjamin P., Cerón, Julián, and Moreno-Mateos, Miguel A.
- Subjects
GENOME editing ,CRISPRS ,NUCLEOPROTEINS ,PLANT cell culture ,CAENORHABDITIS elegans ,ENZYMES ,NUCLEASES - Abstract
The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals. PAM requirement is a constraint for genome editing but this has been circumvented by engineered Cas9 nucleases as SpG and SpRY recognizing minimal PAM sequences. Here, the authors validate and optimize SpG and SpRY in vivo expanding the targeting landscape in animals. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
10. Data from Shanghai Veterinary Research Institute Provide New Insights into Life Science (Riemerella Anatipestifer Endonuclease I Displays Enzymatic Activity and Is Associated With Bacterial Virulence)
- Subjects
Virulence (Microbiology) ,Nucleases ,Enzymes ,Physical fitness ,Health - Abstract
2023 MAY 6 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- Investigators publish new report on Life Science. According to news reporting originating [...]
- Published
- 2023
11. New Enzymes and Coenzymes Study Findings Have Been Reported by a Researcher at Karolinska Institute (ATX-LPA-Dependent Nuclear Translocation of Endonuclease G in Respiratory Epithelial Cells: A New Mode Action for DNA Damage Induced by ...)
- Subjects
DNA ,Nucleases ,DNA damage ,Enzymes ,Physical fitness ,Health - Abstract
2023 FEB 18 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- Researchers detail new data in enzymes and coenzymes. According to news originating [...]
- Published
- 2023
12. A multi-transcriptomics approach reveals the coordinated action of the endoribonuclease DNE1 and the decapping machinery in orchestrating mRNA decay
- Subjects
Machinery ,Arabidopsis thaliana ,Messenger RNA ,Nucleases ,Enzymes ,Magneto-electric machines ,Biological sciences ,Health - Abstract
2024 FEB 20 (NewsRx) -- By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting based on a preprint abstract, our journalists obtained the following [...]
- Published
- 2024
13. Enzymes as Molecular Tools
- Author
-
Valsala, Gayathri, Sugathan, Shiburaj, Sugathan, Shiburaj, editor, Pradeep, N. S., editor, and Abdulhameed, Sabu, editor
- Published
- 2017
- Full Text
- View/download PDF
14. HEPN RNases – an emerging class of functionally distinct RNA processing and degradation enzymes.
- Author
-
Pillon, Monica C., Gordon, Jacob, Frazier, Meredith N., and Stanley, Robin E.
- Subjects
- *
RIBONUCLEASES , *RNA , *ENZYMES , *NUCLEASES , *BACTERIAL evolution , *CRISPRS - Abstract
HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) RNases are an emerging class of functionally diverse RNA processing and degradation enzymes. Members are defined by a small α-helical bundle encompassing a short consensus RNase motif. HEPN dimerization is a universal requirement for RNase activation as the conserved RNase motifs are precisely positioned at the dimer interface to form a composite catalytic center. While the core HEPN fold is conserved, the organization surrounding the HEPN dimer can support large structural deviations that contribute to their specialized functions. HEPN RNases are conserved throughout evolution and include bacterial HEPN RNases such as CRISPR-Cas and toxin-antitoxin associated nucleases, as well as eukaryotic HEPN RNases that adopt large multi-component machines. Here we summarize the canonical elements of the growing HEPN RNase family and identify molecular features that influence RNase function and regulation. We explore similarities and differences between members of the HEPN RNase family and describe the current mechanisms for HEPN RNase activation and inhibition. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
15. A synthetic CRISPR-Cas nuclease with expanded enzymatic activities
- Subjects
Genomics ,Nucleases ,Enzymes ,Biotechnology industry ,Pharmaceuticals and cosmetics industries - Abstract
2023 NOV 29 (NewsRx) -- By a News Reporter-Staff News Editor at Biotech Week -- According to news reporting based on a preprint abstract, our journalists obtained the following quote [...]
- Published
- 2023
16. The Application of a Nuclease Enzyme to Clean Stubborn Soils and Odors in Laundry.
- Author
-
Morales‐Garcia, Ana L., Hayward, Adam S., Malekpour, Adam K., Korzycka, Karolina A., Compson, Rachael, Gori, Klaus, and Lant, Neil J.
- Subjects
- *
LAUNDRY , *SOILS , *ODORS , *ENZYMES , *NUCLEASES - Abstract
The inclusion of a nuclease, a new‐to‐laundry enzyme class in detergent formulations, reduces the concentration of recalcitrant soils on dirty clothing. Nucleases target extracellular DNA that, albeit present in minor quantities in soiled laundry items, disproportionately contributes to the accumulation of sebaceous soils and other compounds that affect the appearance of clothes. These lingering soils accumulate over multiple wear cycles and provoke a dingy appearance in clothing, characterized by dull colors, yellow staining, and an unpleasant malodor. The single and continued use of a nuclease in detergent formulations offers increased removal of these recalcitrant soils to produce less malodorous and visually preferred textiles. Furthermore, the nuclease is effective across different detergent forms, as well as in cotton short washing cycles, enabling improved cleaning in more sustainable laundry washing conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
17. Emergent Properties and Functions of Nanoconfined Nucleic Acid Architectures
- Author
-
Nicholson, Allen W., Redhu, Shiv K., Stopar, Alex, Coral, Lucia, Carnevale, Vincenzo, Castronovo, Matteo, Erdmann, Volker A., editor, Jurga, Stefan, editor, and Barciszewski, Jan, editor
- Published
- 2015
- Full Text
- View/download PDF
18. Researchers from University of Ottawa Publish New Studies and Findings in the Area of Science (An unnatural enzyme with endonuclease activity towards small non-coding RNAs)
- Subjects
Nucleases ,RNA ,Enzymes ,Health ,University of Ottawa - Abstract
2023 JUL 10 (NewsRx) -- By a News Reporter-Staff News Editor at Hepatitis Weekly -- Data detailed on science have been presented. According to news reporting from the University of [...]
- Published
- 2023
19. Study Data from Fudan University Update Understanding of DNA-Directed DNA Polymerase (Intac Endonuclease and Phosphatase Modules Differentially Regulate Transcription By Rna Polymerase Ii)
- Subjects
Cell Press ,DNA ,Genetic research ,Phosphatases ,Nucleases ,RNA ,Enzymes ,Genetic transcription ,Periodical publishing ,Biological sciences ,Health ,Fudan University - Abstract
2023 JUL 4 (NewsRx) -- By a News Reporter-Staff News Editor at Life Science Weekly -- Research findings on Enzymes and Coenzymes - DNA-Directed DNA Polymerase are discussed in a [...]
- Published
- 2023
20. SCIENTIFIC INSTRUMENTS S.A. secures contract for Modifying enzymes of DNA and Endonuclease cloning technologies Specific DNA tolerant to salt degrading the monocatenary DNA
- Subjects
DNA ,Nucleases ,Enzymes ,Cloning ,Contract agreement ,News, opinion and commentary - Abstract
Panama based SCIENTIFIC INSTRUMENTS S.A. has secured contract from INSTITUTO CONMEMORATIVO GORGAS DE ESTUDIOS DE LA SALUD/Compras for Modifying enzymes of DNA and Endonuclease cloning technologies Specific DNA tolerant to [...]
- Published
- 2022
21. Effects of Lyse-It on endonuclease fragmentation, function and activity.
- Author
-
Santaus, Tonya M., Zhang, Fan, Li, Shan, Stine, O. Colin, and Geddes, Chris D.
- Subjects
- *
ENDONUCLEASES , *RIBONUCLEASE A , *POLYACRYLAMIDE gel electrophoresis , *NUCLEIC acids , *NUCLEASES , *DNA-binding proteins - Abstract
Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
22. Characterization of Vibrio cholerae's Extracellular Nuclease Xds.
- Author
-
Pressler, Katharina, Mitterer, Fabian, Vorkapic, Dina, Reidl, Joachim, Oberer, Monika, and Schild, Stefan
- Subjects
VIBRIO cholerae ,CHOLERA ,EXONUCLEASES ,GRAM-negative bacteria ,NUCLEASES ,ENZYMES ,IMMUNE response - Abstract
The Gram-negative bacterium Vibrio cholerae encodes two nucleases, Dns and Xds, which play a major role during the human pathogen's lifecycle. Dns and Xds control three-dimensional biofilm formation and bacterial detachment from biofilms via degradation of extracellular DNA and thus contribute to the environmental, inter-epidemic persistence of the pathogen. During intestinal colonization the enzymes help evade the innate immune response, and therefore promote survival by mediating escape from neutrophil extracellular traps. Xds has the additional function of degrading extracellular DNA down to nucleotides, which are an important nutrient source for V. cholerae. Thus, Xds is a key enzyme for survival fitness during distinct stages of the V. cholerae lifecycle and could be a potential therapeutic target. This study provides detailed information about the enzymatic properties of Xds using purified protein in combination with a real time nuclease activity assay. The data define an optimal buffer composition for Xds activity as 50 mM Tris/HCl pH 7, 100 mM NaCl, 10 mM MgCl
2 , and 20 mM CaCl2 . Moreover, maximal activity was observed using substrate DNA with low GC content and ambient temperatures of 20–25°C. In silico analysis and homology modeling predicted an exonuclease domain in the C-terminal part of the protein. Biochemical analyses with truncated variants and point mutants of Xds confirm that the C-terminal region is sufficient for nuclease activity. We also find that residues D787 and H837 within the predicted exonuclease domain are key to formation of the catalytic center. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
23. Nucleotide substrate binding characterization in human pancreatic-type ribonucleases.
- Author
-
Bafna, Khushboo, Narayanan, Chitra, Chennubhotla, S. Chakra, Doucet, Nicolas, and Agarwal, Pratul K.
- Subjects
- *
MOLECULAR biology , *REVERSE transcriptase , *LIFE sciences , *PHYSICAL sciences - Abstract
Human genome contains a group of more than a dozen similar genes with diverse biological functions including antiviral, antibacterial and angiogenesis activities. The characterized gene products of this group show significant sequence similarity and a common structural fold associated with binding and cleavage of ribonucleic acid (RNA) substrates. Therefore, these proteins have been categorized as members of human pancreatic-type ribonucleases (hRNases). hRNases differ in cell/tissue localization and display distinct substrate binding preferences and a wide range of ribonucleolytic catalytic efficiencies. Limited information is available about structural and dynamical properties that influence this diversity among these homologous RNases. Here, we use computer simulations to characterize substrate interactions, electrostatics and dynamical properties of hRNases 1–7 associated with binding to two nucleotide substrates (ACAC and AUAU). Results indicate that even with complete conservation of active-site catalytic triad associated with ribonucleolytic activity, these enzymes show significant differences in substrate interactions. Detailed characterization suggests that in addition to binding site electrostatic and van der Waals interactions, dynamics of distal regions may also play a role in binding. Another key insight is that a small difference in temperature of 300 K (used in experimental studies) and 310 K (physiological temperature) shows significant changes in enzyme-substrate interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
24. Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels.
- Author
-
Simsa, Robin, Vila, Xavier Monforte, Salzer, Elias, Teuschl, Andreas, Jenndahl, Lachmi, Bergh, Niklas, and Fogelstrand, Per
- Subjects
- *
FLUID dynamics , *BLOOD vessels , *EXTRACELLULAR matrix proteins , *VASCULAR grafts , *TRIBUTYL phosphate - Abstract
Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
25. Involvement of PIN‐like domain nucleases in tRNA processing and translation regulation.
- Author
-
Gobert, Anthony, Bruggeman, Mathieu, and Giegé, Philippe
- Subjects
- *
TRANSFER RNA , *NUCLEASES , *RIBONUCLEASES , *ENZYMES , *TRANSLATIONS , *PROTEINS - Abstract
Transfer RNAs require essential maturation steps to become functional. Among them, RNase P removes 5′ leader sequences of pre‐tRNAs. Although RNase P was long thought to occur universally as ribonucleoproteins, different types of protein‐only RNase P enzymes were discovered in both eukaryotes and prokaryotes. Interestingly, all these enzymes belong to the super‐group of PilT N‐terminal‐like nucleases (PIN)‐like ribonucleases. This wide family of enzymes can be subdivided into major subgroups. Here, we review recent studies at both functional and mechanistic levels on three PIN‐like ribonucleases groups containing enzymes connected to tRNA maturation and/or translation regulation. The evolutive distribution of these proteins containing PIN‐like domains as well as their organization and fusion with various functional domains is discussed and put in perspective with the diversity of functions they acquired during evolution, for the maturation and homeostasis of tRNA and a wider array of RNA substrates. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1117–1125, 2019 [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
26. Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.
- Author
-
Camerlingo, Rosa, Miceli, Roberta, Marra, Laura, Rea, Giuseppina, D’Agnano, Igea, Nardella, Marta, Montella, Roberta, Morabito, Alessandro, Normanno, Nicola, Tirino, Virginia, and Rocco, Gaetano
- Subjects
- *
OVARIAN follicle , *LUNG cancer , *CANCER cells , *NON-small-cell lung carcinoma , *CELL lines , *CANCER invasiveness - Abstract
The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-β1 quantification. We found that LC212-CM contains high levels of TGF-β1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-β1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-β1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-β1. In conclusion, hsa-miR21 and TGF-β1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
27. Oil degradation potential of microbial communities in water and sediment of Baltic Sea coastal area.
- Author
-
Miettinen, Hanna, Bomberg, Malin, Nyyssönen, Mari, Reunamo, Anna, Jørgensen, Kirsten S., and Vikman, Minna
- Subjects
- *
RIBOSOMAL RNA , *MICROBIAL communities , *MATERIALS science , *EARTH sciences , *FUNGAL genes , *PHYSICAL sciences - Abstract
Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH–RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
28. Energetic costs of cellular and therapeutic control of stochastic mitochondrial DNA populations.
- Author
-
Hoitzing, Hanne, Gammage, Payam A., Haute, Lindsey van, Minczuk, Michal, Johnston, Iain G., and Jones, Nick S.
- Subjects
- *
MITOCHONDRIAL DNA , *BOTANY , *CYTOLOGY , *GENE therapy , *PHYSICAL sciences , *MOLECULAR biology - Abstract
The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
29. A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact.
- Author
-
Kusmierek, Maria, Hoßmann, Jörn, Witte, Rebekka, Opitz, Wiebke, Vollmer, Ines, Volk, Marcel, Heroven, Ann Kathrin, Wolf-Watz, Hans, and Dersch, Petra
- Subjects
- *
GRAM-negative bacteria , *PSEUDOTUBERCULOSIS , *NUCLEIC acids , *POLYMERASE chain reaction , *PHYSIOLOGY - Abstract
Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
30. Comment on "A comprehensive overview and evaluation of circular RNA detection tools".
- Author
-
Chen, Chia-Ying and Chuang, Trees-Juen
- Subjects
- *
CIRCULAR RNA , *NON-coding RNA , *COMPUTATIONAL biology - Abstract
A review of the article "A comprehensive overview and evaluation of circular RNA detection tools" which appeared in a previous issue of the periodical "PLOS Computational Biology" is presented.
- Published
- 2019
- Full Text
- View/download PDF
31. Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.
- Author
-
Deasy, Sarah K., Uehara, Ryo, Vodnala, Suman K., Yang, Howard H., Dass, Randall A., Hu, Ying, Lee, Maxwell P., Crouch, Robert J., and Hunter, Kent W.
- Subjects
- *
BREAST cancer , *HAPLOTYPES , *CANCER invasiveness , *IMMUNE response , *IMMUNOPHENOTYPING - Abstract
Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
32. An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability.
- Author
-
Dorn, Annika, Feller, Laura, Castri, Dominique, Röhrig, Sarah, Enderle, Janina, Herrmann, Natalie J., Block-Schmidt, Astrid, Trapp, Oliver, Köhler, Laura, and Puchta, Holger
- Subjects
- *
ARABIDOPSIS , *FANCONI'S anemia , *DNA analysis , *CELL death , *ENZYMES - Abstract
Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
33. Details in the evaluation of circular RNA detection tools: Reply to Chen and Chuang.
- Author
-
Zeng, Xiangxiang, Lin, Wei, Guo, Maozu, and Zou, Quan
- Subjects
- *
CIRCULAR RNA , *BIG data , *TOXINS , *PLASMIDS , *DATABASES - Abstract
In their comment, Chen and Chuang [] pointed out several weak points of our recent paper []. Here we respond in detail to clarify the dataset we used in our work. We also discuss the three confounding factors they listed in their comment. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
34. Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.
- Author
-
Ota, Yuri, Saito, Kanako, Takagi, Taeko, Matsukura, Satoko, Morita, Masamune, Tsuneda, Satoshi, and Noda, Naohiro
- Subjects
- *
NUCLEIC acid probes , *BACTERIAL growth , *FLUORESCENT probes , *FLUORESCENCE resonance energy transfer , *DROPLETS , *GROWTH rate - Abstract
We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
35. Transcription is a major driving force for plastid genome instability in Arabidopsis.
- Author
-
Pérez Di Giorgio, Juliana Andrea, Lepage, Étienne, Tremblay-Belzile, Samuel, Truche, Sébastien, Loubert-Hudon, Audrey, and Brisson, Normand
- Subjects
- *
GENE rearrangement , *COMPLEMENTARY DNA , *ARABIDOPSIS , *POLYMERASES , *BOTANY , *PLANT genetics - Abstract
Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
36. Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing.
- Author
-
Ignatov, Konstantin B., Blagodatskikh, Konstantin A., Shcherbo, Dmitry S., Kramarova, Tatiana V., Monakhova, Yulia A., and Kramarov, Vladimir M.
- Subjects
- *
NUCLEOTIDE sequence , *NUCLEIC acids , *SINGLE-stranded DNA , *POLYMERIZATION - Abstract
Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
37. Elimination of bacterial DNA during RNA isolation from sputum: Bashing bead vortexing is preferable over prolonged DNase treatment.
- Author
-
Paska, Csilla, Barta, Imre, Drozdovszky, Orsolya, and Antus, Balazs
- Subjects
- *
DNA primers , *BACTERIAL DNA , *NUCLEIC acid isolation methods , *RNA , *SPUTUM , *OBSTRUCTIVE lung diseases - Abstract
Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
38. Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not.
- Author
-
Pons, Benoît J., Bezine, Elisabeth, Hanique, Mélissa, Guillet, Valérie, Mourey, Lionel, Chicher, Johana, Frisan, Teresa, Vignard, Julien, and Mirey, Gladys
- Subjects
- *
EUKARYOTIC cells , *DNA damage , *TOXINS , *MOLECULAR biology - Abstract
The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays–the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
39. Apolipoprotein E-C1-C4-C2 gene cluster region and inter-individual variation in plasma lipoprotein levels: a comprehensive genetic association study in two ethnic groups.
- Author
-
Pirim, Dilek, Radwan, Zaheda H., Wang, Xingbin, Niemsiri, Vipavee, Hokanson, John E., Hamman, Richard F., Feingold, Eleanor, Bunker, Clareann H., Demirci, F. Yesim, and Kamboh, M. Ilyas
- Subjects
- *
CHOLESTERYL ester transfer protein , *GENE clusters , *ETHNIC studies , *BLOOD lipids , *ETHNIC groups , *MOLECULAR biology - Abstract
The apolipoprotein E-C1-C4-C2 gene cluster at 19q13.32 encodes four amphipathic apolipoproteins. The influence of APOE common polymorphisms on plasma lipid/lipoprotein profile, especially on LDL-related traits, is well recognized; however, little is known about the role of other genes/variants in this gene cluster. In this study, we evaluated the role of common and uncommon/rare genetic variation in this gene region on inter-individual variation in plasma lipoprotein levels in non-Hispanic Whites (NHWs) and African blacks (ABs). In the variant discovery step, the APOE, APOC1, APOC4, APOC2 genes were sequenced along with their flanking and hepatic control regions (HCR1 and HCR2) in 190 subjects with extreme HDL-C/TG levels. The next step involved the genotyping of 623 NHWs and 788 ABs for the identified uncommon/rare variants and common tagSNPs along with additional relevant SNPs selected from public resources, followed by association analyses with lipid traits. A total of 230 sequence variants, including 15 indels were identified, of which 65 were novel. A total of 70 QC-passed variants in NHWs and 108 QC-passed variants in ABs were included in the final association analyses. Single-site association analysis of SNPs with MAF>1% revealed 20 variants in NHWs and 24 variants in ABs showing evidence of association with at least one lipid trait, including several variants exhibiting independent associations from the established APOE polymorphism even after multiple-testing correction. Overall, our study has confirmed known associations and also identified novel associations in this genomic region with various lipid traits. Our data also support the contribution of both common and uncommon/rare variation in this gene region in affecting plasma lipid profile in the general population. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
40. Aicardi–Goutières Syndrome associated mutations of RNase H2B impair its interaction with ZMYM3 and the CoREST histone-modifying complex.
- Author
-
Shapson-Coe, Alexander, Valeiras, Brenda, Wall, Christopher, and Rada, Cristina
- Subjects
- *
ZINC-finger proteins , *SCAFFOLD proteins - Abstract
DNA-RNA hybrids arise in all cell types, and are removed by multiple enzymes, including the trimeric ribonuclease, RNase H2. Mutations in human RNase H2 result in Aicardi–Goutières syndrome (AGS), an inflammatory brain disorder notable for being a Mendelian mimic of congenital viral infection. Previous studies have shown that several AGS-associated mutations of the RNase H2B subunit do not affect trimer stability or catalytic activity and are clustered on the surface of the complex, leading us to speculate that these mutations might impair important interactions of RNase H2 with so far unidentified proteins. In this study, we show that AGS mutations in this cluster impair the interaction of RNase H2 with several members of the CoREST chromatin-silencing complex that include the histone deacetylase HDAC2 and the demethylase KDM1A, the transcriptional regulators RCOR1 and GTFII-I as well as ZMYM3, an MYM-type zinc finger protein. We also show that the interaction is mediated by the zinc finger protein ZMYM3, suggesting that ZMYM3 acts as a novel type of scaffold protein coordinating interactions between deacetylase, demethylase and RNase H type enzymes, raising the question of whether coordination between histone modifications and the degradation of RNA-DNA hybrids may be required to prevent inflammation in humans. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
41. Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.
- Author
-
Pakkulnan, Rattiyaphorn, Anutrakunchai, Chitchanok, Kanthawong, Sakawrat, Taweechaisupapong, Suwimol, Chareonsudjai, Pisit, and Chareonsudjai, Sorujsiri
- Subjects
- *
BURKHOLDERIA pseudomallei , *BACTERIAL adhesion , *BACTERIAL DNA , *BIOMASS , *LIFE sciences , *GENTIAN violet - Abstract
The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
42. The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.
- Author
-
Pennington, Helen G., Jones, Rhian, Kwon, Seomun, Bonciani, Giulia, Thieron, Hannah, Chandler, Thomas, Luong, Peggy, Morgan, Sian Natasha, Przydacz, Michal, Bozkurt, Tolga, Bowden, Sarah, Craze, Melanie, Wallington, Emma J., Garnett, James, Kwaaitaal, Mark, Panstruga, Ralph, Cota, Ernesto, and Spanu, Pietro D.
- Subjects
- *
ERYSIPHE graminis , *POWDERY mildew diseases , *GRAIN diseases & pests , *RIBOSOMAL RNA , *RIBONUCLEASES - Abstract
The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
43. Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering.
- Author
-
Raitskin, Oleg, Schudoma, Christian, West, Anthony, and Patron, Nicola J.
- Subjects
- *
CRISPRS , *NUCLEASES , *PLANT genomes , *PLANT mutation , *PLANT genetic engineering - Abstract
Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
44. A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins.
- Author
-
Hagen, Benedikt, Kraase, Martin, Indikova, Ivana, and Indik, Stanislav
- Subjects
- *
MOUSE mammary tumor virus , *POLYMERIZATION , *RETROVIRUSES , *ONCOGENIC viruses , *REVERSE transcriptase , *DNA polymerases - Abstract
Retroviruses have evolved multiple means to counteract host restriction factors such as single-stranded DNA-specific deoxycytidine deaminases (APOBEC3s, A3s). These include exclusion of A3s from virions by an A3-unreactive nucleocapsid or expression of an A3-neutralizing protein (Vif, Bet). However, a number of retroviruses package A3s and do not encode apparent vif- or bet-like genes, yet they replicate in the presence of A3s. The mode by which they overcome deleterious restriction remains largely unknown. Here we show that the prototypic betaretrovirus, mouse mammary tumor virus (MMTV), packages similar amounts of A3s as HIV-1ΔVif, yet its proviruses carry a significantly lower level of A3-mediated deamination events than the lentivirus. The G-to-A mutation rate increases when the kinetics of reverse transcription is reduced by introducing a mutation (F120L) to the DNA polymerase domain of the MMTV reverse transcriptase (RT). A similar A3-sensitizing effect was observed when the exposure time of single-stranded DNA intermediates to A3s during reverse transcription was lengthened by reducing the dNTP concentration or by adding suboptimal concentrations of an RT inhibitor to infected cells. Thus, the MMTV RT has evolved to impede access of A3s to transiently exposed minus DNA strands during reverse transcription, thereby alleviating inhibition by A3 family members. A similar mechanism may be used by other retroviruses and retrotransposons to reduce deleterious effects of A3 proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
45. mRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.
- Author
-
Ben-Zvi, Tamar, Pushkarev, Alina, Seri, Hemda, Elgrably-Weiss, Maya, Papenfort, Kai, and Altuvia, Shoshy
- Subjects
- *
MESSENGER RNA , *PUTRESCINE , *POLYAMINES , *BIOSYNTHESIS , *ARGININE , *SALMONELLA - Abstract
Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
46. Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing.
- Author
-
Kim, Eung Kweon, Kim, Seunghyuk, and Maeng, Yong-Sun
- Subjects
- *
DYSTROPHY , *STEM cells , *CRISPRS , *GENOME editing , *POINT mutation (Biology) , *CORNEAL dystrophies - Abstract
Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
47. The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets.
- Author
-
Loza-Correa, Maria, Ayala, Juan A., Perelman, Iris, Hubbard, Keith, Kalab, Miloslav, Yi, Qi-Long, Taha, Mariam, de Pedro, Miguel A., and Ramirez-Arcos, Sandra
- Subjects
- *
PEPTIDOGLYCANS , *STAPHYLOCOCCUS epidermidis , *BLOOD platelets , *IMMUNE system , *IMMUNE response , *BIOFILMS , *ENVIRONMENTAL engineering - Abstract
Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
48. RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.
- Author
-
Posse, Viktor, Al-Behadili, Ali, Uhler, Jay P., Clausen, Anders R., Reyes, Aurelio, Zeviani, Massimo, Falkenberg, Maria, and Gustafsson, Claes M.
- Subjects
- *
HUMAN mitochondrial DNA , *DNA replication , *RIBONUCLEASES , *DNA synthesis , *GENETIC transcription - Abstract
Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
49. A novel variant of torque teno virus 7 identified in patients with Kawasaki disease.
- Author
-
Thissen, James B., Isshiki, Mariko, Jaing, Crystal, Nagao, Yoshiro, Lebron Aldea, Dayanara, Allen, Jonathan E., Izui, Masafumi, Slezak, Thomas R., Ishida, Takafumi, and Sano, Tetsuya
- Subjects
- *
TORQUE teno virus , *MUCOCUTANEOUS lymph node syndrome , *ETIOLOGY of diseases , *EPIDEMIOLOGY ,CORONARY artery abnormalities - Abstract
Kawasaki disease (KD), first identified in 1967, is a pediatric vasculitis of unknown etiology that has an increasing incidence in Japan and many other countries. KD can cause coronary artery aneurysms. Its epidemiological characteristics, such as seasonality and clinical picture of acute systemic inflammation with prodromal intestinal/respiratory symptoms, suggest an infectious etiology for KD. Interestingly, multiple host genotypes have been identified as predisposing factors for KD. To explore experimental methodology for identifying etiological agent(s) for KD and to optimize epidemiological study design (particularly the sample size) for future studies, we conducted a pilot study. For a 1-year period, we prospectively enrolled 11 patients with KD. To each KD patient, we assigned two control individuals (one with diarrhea and the other with respiratory infections), matched for age, sex, and season of diagnosis. During the acute phase of disease, we collected peripheral blood, nasopharyngeal aspirate, and feces. We also determined genotypes, to identify those that confer susceptibility to KD. There was no statistically significant difference in the frequency of the risk genotypes between KD patients and control subjects. We also used unbiased metagenomic sequencing to analyze these samples. Metagenomic sequencing and PCR detected torque teno virus 7 (TTV7) in two patients with KD (18%), but not in control subjects (P = 0.111). Sanger sequencing revealed that the TTV7 found in the two KD patients contained almost identical variants in nucleotide and identical changes in resulting amino acid, relative to the reference sequence. Additionally, we estimated the sample size that would be required to demonstrate a statistical correlation between TTV7 and KD. Future larger scale studies with carefully optimized metagenomic sequencing experiments and adequate sample size are warranted to further examine the association between KD and potential pathogens, including TTV7. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
50. Plasmodium apicoplast tyrosyl-tRNA synthetase recognizes an unusual, simplified identity set in cognate tRNATyr.
- Author
-
Cela, Marta, Paulus, Caroline, Santos, Manuel A. S., Moura, Gabriela R., Frugier, Magali, and Rudinger-Thirion, Joëlle
- Subjects
- *
PLASMODIUM , *TRANSFER RNA synthetases , *CYTOSOL , *BIOSYNTHESIS , *BIOCHEMICAL substrates - Abstract
The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.