Bifunctional Sucrose Phosphate Synthasephosphatase Is Involved In
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Comparative Genomics Revealing Insights Into Niche Separation of the Genus Methylophilus
microorganisms Article Comparative Genomics Revealing Insights into Niche Separation of the Genus Methylophilus Nana Lin 1,2, Ye Tao 2,3, Peixin Gao 2, Yan Xu 1 and Peng Xing 2,* 1 School of Civil Engineering, Southeast University, Nanjing 210096, China; [email protected] (N.L.); [email protected] (Y.X.) 2 State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China; [email protected] (Y.T.); [email protected] (P.G.) 3 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected]; Tel.: +86-25-8688-2112; Fax: +86-25-5771-4759 Abstract: The genus Methylophilus uses methanol as a carbon and energy source, which is widely distributed in terrestrial, freshwater and marine ecosystems. Here, three strains (13, 14 and QUAN) related to the genus Methylophilus, were newly isolated from Lake Fuxian sediments. The draft genomes of strains 13, 14 and QUAN were 3.11 Mb, 3.02 Mb, 3.15 Mb with a G+C content of 51.13, 50.48 and 50.33%, respectively. ANI values between strains 13 and 14, 13 and QUAN, and 14 and QUAN were 81.09, 81.06 and 91.46%, respectively. Pan-genome and core-genome included 3994 and 1559 genes across 18 Methylophilus genomes, respectively. Phylogenetic analysis based on 1035 single-copy genes and 16S rRNA genes revealed two clades, one containing strains isolated from aquatic and the other from the leaf surface. Twenty-three aquatic-specific genes, such as 2OG/Fe(II) oxygenase and diguanylate cyclase, reflected the strategy to survive in oxygen-limited water and sediment. -
RT-PCR Protocol Utilizing RT-PCR
Protocol TD-P Revision 1.0 Creation Date: 2/18/2019 Revision Date: 4/24/2019 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Utilizing RT-PCR Kit Introduction Our RT-PCR Kit combines two powerful mixtures: i) 5x Master Mix and ii) Hot start Taq 2x master mix with a standard buffer for 2-step RT-PCR. The two mixtures require minimal handling during the reaction setup and offer consistent and robust RT-PCR reactions. First strand cDNA synthesis is achieved by using the 5x Master Mix, which contains Reverse Transcriptase, recombinant RNase inhibitor, dNTPs, an optimized buffer, MgCl2 and protein stabilizers. Reverse Transcriptase is a recombinant MMLV reverse transcriptase with reduced RNase H activity and increased thermostability. This kit also provides two optimized primers and nuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail and the random hexamer primer mix provides random and consistent priming sites covering the entire RNA templates including both mRNAs and non- polyadenylated RNAs. In addition, this kit is highly efficient at producing full-length cDNA from long RNA templates at temperatures between 42-55°C. The amplification step features a high quality Hot Start Taq DNA Polymerase, which offers higher fidelity and better amplification in a master mix format. RT-PCR product can be generated up to 6 kb. Our RT-PCR Kit can be used for cDNA synthesis followed by gene expression data validation. The advantages of our RT-PCR Kit include robust and active cDNA synthesis at temperatures up to 55°C and high efficiency at producing full-length cDNA from as little as 50 pg of total RNA. -
Pfu DNA Polymerase Deoxynucleoside-Triphosphate: DNA Deoxynucleotidyl-Transferase (DNA-Directed); EC 2.7.7.7
Version 060918 G:\products\productflyer\pcr\polymerasen\proof\descr_m3004_pfu_en.docx ENAXXON fon: +49(0)731 – 3608 123 X G fax: +49(0)731 – 3608 962 b i o s c i e n c e E-Mail: [email protected] Pfu DNA Polymerase Deoxynucleoside-triphosphate: DNA deoxynucleotidyl-transferase (DNA-directed); EC 2.7.7.7 Product Cat# Package size Pfu DNA Polymerase (proof-reading polymerase) M3004.0250 250 units Pfu DNA Polymerase (proof-reading polymerase) M3004.0500 2 x 250 units Pfu DNA Polymerase (proof-reading polymerase) M3004.1250 5 x 250 units Description The Genaxxon bioscience Pfu DNA Polymerase is a thermostable enzyme possessing 5‘-3‘ DNA polymerase and 3‘-5‘ proof reading exonuclease activities. It is isolated from the hyperthermophilic marine archae Pyroccocus furiosus (Pfu). The enzyme provides extremely high fidelity. Whereas the enzyme is not able to amplify long fragments as efficiently as the Genaxxon Taq polymerase because of its very high exonuclease activity. To overcome this restriction we recommend to use the Genaxxon ReproFast DNA Polymerase, which will provide a more robust synthesis of longer amplification products (Barnes et al. (1994) Proc. Natl. Acad. Sci., USA 91, 2216-2220). Use of the Genaxxon Pfu DNA Polymerase in amplification results in blunt-ended products, which is not recommended for cloning into T/A vectors. Concentration: 2.5 units/µL Unit definition: One unit is defined as the amount of enzyme that incorporates 10nmoles of dNTPs into acid-insoluble form in 30 min at 72°C under the assay conditions (25mM TAPS (tris-(hydroxymethyl)methyl-amino-propane-sulphonic acid, sodium salt) pH9.3 (at 25°C), 50mM KCl, 2mM MgCl2, 1mM ß-mercaptoethanol) and activated calf thymus DNA as substrate. -
Biotechnology Explorer™
Biotechnology Explorer™ GAPDH PCR Module Instruction Manual Catalog #166-5010EDU explorer.bio-rad.com This kit is shipped at 4°C. Open immediately upon arrival and store reagents at –20°C within 2 weeks. Duplication of any part of this document permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Biotechnology Explorer kit curricula. For technical support, call your local Bio-Rad office or, in the U.S., call 1-800-424-6723 Dear Educator: Amplification as the path to visualization of DNA Molecular biologists are faced with the classic needle in a haystack problem. Often we must find a few copies of a piece of DNA that code for a given gene in the haystack of DNA comprising the entire genome. Even if there are a thousand copies of the same piece of DNA it is often still difficult to locate them. However by selectively amplifying only that specific piece of DNA we can separate it from the rest of the DNA and visualize it. Once we can visualize the DNA, we can use the tools of molecular biology to open the whole vista of genetic engineering. We can work with the DNA to make discoveries in science, agriculture, and medicine. The method used to amplify the DNA is the polymerase chain reaction (PCR). PCR is capable of repeatedly doubling the amount of specific DNA. After many PCR cycles a million-fold or billion-fold times as much DNA is generated. Because of the increasing use of PCR in science it is important to provide students with an understanding of the basic principles and applications of PCR. -
The Heterodimeric Primase from the Euryarchaea Pyrococcus Abyssi: A
Journal of Molecular Biology Archimer, archive institutionnelle de l’Ifremer Article in Press http://www.ifremer.fr/docelec/ Acceptation date : 5 October 2007 http://dx.doi.org/10.1016/j.jmb.2007.10.015 © 2007 Elsevier ailable on the publisher Web site The heterodimeric primase from the Euryarchaea Pyrococcus abyssi: a multifunctional enzyme for initiation and repair ? Magali Le Breton1, Ghislaine Henneke1, Cédric Norais2, Didier Flament1, Hannu Myllykallio2, Joël Querellou1 and Jean-Paul Raffin1,∗. 1. Ifremer, UMR6197, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, F-29280 blisher-authenticated version is av Plouzané, France. 2. Institut de Génétique et Microbiologie, UMR8621, Université Paris-sud, 91405 Orsay, France ∗Corresponding author: Jean Paul Raffin, Ifremer, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, 29280 Plouzané, France. Phone: 33(0)2 98 22 45 39 ; [email protected] Abstract: We report the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of two proteins, Pabp41 and Pabp46, showing sequence similarities respectively to the p49 and p58 subunits of the eukaryotic polymerase α-primase complex. Both subunits were expressed, purified and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribo- and ccepted for publication following peer review. The definitive pu deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. -
C1 Compounds Shape the Microbial Community of an Abandoned Century-Old Oil
bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278820; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. C1 compounds shape the microbial community of an abandoned century-old oil exploration well. 1 Diego Rojas-Gätjens1, Paola Fuentes-Schweizer2,3, Keilor Rojas-Jimenez,4, Danilo Pérez-Pantoja5, Roberto 2 Avendaño1, Randall Alpízar6, Carolina Coronado-Ruíz1 & Max Chavarría1,3,7* 3 4 1Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), CeNAT-CONARE, 1174-1200 San José 5 (Costa Rica). 2Centro de Investigación en electroquímica y Energía química (CELEQ), Universidad de 6 Costa Rica, 11501-2060 San José (Costa Rica). 3Escuela de Química, Universidad de Costa Rica, 11501- 7 2060 San José (Costa Rica). 4Escuela de Biología, Universidad de Costa Rica, 11501-2060 San José 8 (Costa Rica). 5Programa Institucional de Fomento a la Investigación, Desarrollo, e Innovación (PIDi), 9 Universidad Tecnológica Metropolitana, Santiago (Chile). 6Hidro Ambiente Consultores, 202, San José 10 (Costa Rica), 7Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, 11 11501-2060 San José (Costa Rica). 12 Keywords: Methylotrophic bacteria, Methylobacillus, Methylococcus, Methylorubrum, Hydrocarbons, 13 Oil well, Methane, Cahuita National Park 14 *Correspondence to: Max Chavarría 15 Escuela de Química & Centro de Investigaciones en Productos Naturales (CIPRONA) 16 Universidad de Costa Rica 17 Sede Central, San Pedro de Montes de Oca 18 San José, 11501-2060, Costa Rica 19 Phone (+506) 2511 8520. Fax (+506) 2253 5020 20 E-mail: [email protected] 21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278820; this version posted September 2, 2020. -
SIP Metagenomics Identifies Uncultivated Methylophilaceae As Dimethylsulphide Degrading Bacteria in Soil and Lake Sediment
SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment. Eyice, Ö; Namura, M; Chen, Y; Mead, A; Samavedam, S; Schäfer, H http://www.nature.com/ismej/journal/v9/n11/full/ismej201537a.html doi:10.1038/ismej.2015.37 For additional information about this publication click this link. http://qmro.qmul.ac.uk/xmlui/handle/123456789/9962 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] The ISME Journal (2015) 9, 2336–2348 © 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 OPEN www.nature.com/ismej ORIGINAL ARTICLE SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment Özge Eyice1, Motonobu Namura2, Yin Chen1, Andrew Mead1,3, Siva Samavedam1 and Hendrik Schäfer1 1School of Life Sciences, University of Warwick, Coventry, UK and 2MOAC Doctoral Training Centre, University of Warwick, Coventry, UK Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles in situ. In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). -
High-Fidelity Amplification Using a Thermostable DNA Polymerase Isolated from P’~Ococcusfuriosus
Gette, 108 (1991) 1-6 8 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/SO3.50 GENE 06172 High-fidelity amplification using a thermostable DNA polymerase isolated from P’~OCOCCUSfuriosus (Polymerase chain reaction; mutation frequency; lack; proofreading; 3’40-5 exonuclease; recombinant DNA; archaebacteria) Kelly S. Lundberg”, Dan D. Shoemaker*, Michael W.W. Adamsb, Jay M. Short”, Joseph A. Serge* and Eric J. Mathur” “ Division of Research and Development, Stratagene, Inc., La Jolla, CA 92037 (U.S.A.), and h Centerfor Metalloen~~vme Studies, University of Georgia, Athens, GA 30602 (U.S.A.) Tel. (404/542-2060 Received by M. Salas: 16 May 1991 Revised/Accepted: 11 August/l3 August 1991 Received at publishers: 17 September 1991 -.-- SUMMARY A thermostable DNA polymerase which possesses an associated 3’-to-5’ exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10s.~ of the number of mutations obtained from similar amplifications performed with Tuq DNA polymerase. The PCR fidelity assay is based on the ampli~cation and cloning of lad, lac0 and IacZor gene sequences (IcrcIOZa) using either ffil or 7ii~irqDNA poIymerase. Certain mutations within the lucf gene inactivate the Lac repressor protein and permit the expression of /?Gal. When plated on a chromogenic substrate, these Lacl - mutants exhibit a blue-plaque phenotype. -
Pfu DNA Polymerase
344PR-01 G-Biosciences, St Louis, MO. USA ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected] A Geno Technology, Inc. (USA) brand name Pfu DNA Polymerase INTRODUCTION Pfu DNA polymerase, derived from the hyperthermophilic archae Pyrococcus furiosus, has superior thermostability and proofreading properties compared to other thermostable polymerase. Its molecular weight is 90 kD. It can amplify DNA target up to 2kb. The elongation velocity is 0.2~0.4kb/min (70~75°C). Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. Using Pfu DNA polymerase in your PCR reactions results in blunt-ended PCR products, which are ideal for cloning into blunt-ended vectors. Pfu DNA polymerase is superior for techniques that require high-fidelity DNA synthesis. ITEM(S) SUPPLIED Cat. # 786-816 Pfu DNA Polymerase (2.5U/µl) 500U 10X Pfu Buffer (Mg2+ plus) 2 x 1.4ml 6X Loading Buffer 1ml STORAGE CONDITIONS The kit is shipped at ambient temperature. Upon arrival, store at -20oC. Storage buffer is 20mM Tris.HCl (pH8.0), 100mM KCl, 3mM MgCl2, 1mM DTT, 0.1% Nonidet® P-40, 0.1% Tween® 20, 0.2mg/ml BSA, 50% (v/v) glycerol. 10X Pfu BUFFER 200mM Tris-HCl (pH8.8), 100mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton® X-100 and 1mg/ml BSA. UNIT DEFINITION One unit (U) of Pfu polymerase is defined as the amount of enzyme needed to catalyze the incorporation of 10 nanomoles of deoxyribonucleotides into acid-insoluble material in 30 minutes at 70°C using herring sperm DNA as a substrate. -
High-Level Expression, Purification, and Thermus Aquatlcus DNA
Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquatlcus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity Frances C. Lawyer, 1 Susanne Stoffel, 1 Randall K. Saiki, 2 Sheng-Yung Chang, 3 Phoebe A. Landre, ~ Richard D. Abrarnson, 1 and David H. Gelfand 1 1Program in Core Research and Departments of 2Human Genetics and 3Infectious Disease, Roche Molecular Systems, Alameda, California 94501 The Thermus aquaticus DNA poly- life of 9 min at 97.5~ The Stoffel I in the native host is quite low (0.01- merase I (Taq Pol I) gene was cloned fragment has a half-life of 21 min at 0.02% of total protein). The cloning and into a plasmid expression vector that 97.5~ Taq Pol I contains a polymer- expression of full-length 94-kD Taq Pol I utilizes the strong bacteriophage ization-dependent 5' to 3' exonu- in E. coli under control of the E. coli lac PL promoter. A truncated form of Taq clease activity whereas the Stoffel promoter r or the tac promoter (7~ has Pol I was also constructed. The two fragment, deleted for the 5' to 3' ex- been reported. Because polymerase constructs made it possible to com- onuclease domain, does not possess yields in these constructs were low pare the full-length 832-amino-acid that activity. A comparison is made (-0.01% of total protein in our initial Taq Pol I and a deletion derivative among thermostable DNA poly- construct; see ref. -
BIOTOOLS Pfu DNA POLYMERASE (1 U/Μl)
WARRANTY Products are guaranteed to conform to the quality and content indicated on each vial and external labels during their shelf life. BIOTOOLS obligation and purchaser’s rights under this warranty are limited to the replacement by BIOTOOLS of any product that is shown defective in fabrication, and that must be returned to BIOTOOLS, freight prepaid, or at BIOTOOLS’ option, replacement of the purchasing price. Any complaint on damaged goods during transport must be directed to the handling or transport agent. Product for Research Use Only. This product must be used by qualified professionals only. It is the responsibility of the user to ascertain that a given product is adequate for a given application. Any product not fulfilling the specifications included in the product sheet will be replaced. This warranty limits our responsibility to the replacement of the product. No other warranties, of any kind, express or implied, including, without limitation, implicit warranties of commercialisation ability or adequacy for a given BIOTOOLS Pfu DNA POLYMERASE (1 U/l) purpose, are provided by BIOTOOLS. BIOTOOLS will not be held responsible for any direct, indirect, consequential or incidental damage resulting of the use, misuses, results of the use or inability to use any product. Produced by: REF. FORMAT CONTENT BIOTOOLS, Biotechnological & Medical Laboratories, S.A. have been evaluated and certified to accomplish ISO 9001:2000 requirements for the following activities: Research and development of biotechnology products and manufacture of biotechnology and in vitro products. Valle de Tobalina – 52 – Nave 39, 28021 Madrid – Spain BIOTOOLS Pfu DNA Polymerase (1 U/µl) 10.501 100 U © 2008 BIOTOOLS, Biotechnological & Medical Laboratories, S.A. -
The Human TTAGGG Repeat Factors 1 and 2 Bind to a Subset of Interstitial Telomeric Sequences and Satellite Repeats
npg Extratelomeric binding of TRF1 and TRF2 Cell Research (2011) 21:1028-1038. 1028 © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats Thomas Simonet1, Laure-Emmanuelle Zaragosi2, Claude Philippe3, Kevin Lebrigand2, Clémentine Schouteden1, Adeline Augereau1, 3, Serge Bauwens1, Jing Ye1, 3, Marco Santagostino4, Elena Giulotto4, Frederique Magdinier1, Béatrice Horard1, Pascal Barbry2, Rainer Waldmann2, Eric Gilson1, 3, 5 1Laboratoire de Biologie Moléculaire de la Cellule-UMR 5239 CNRS/ENS Lyon/ Université Lyon, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, Lyon 69364, France; 2CNRS and University of Nice Sophia Antipolis, Institut de Pharmacologie Molé- culaire et Cellulaire, 06560 Sophia Antipolis, France; 3Laboratory of Biology and Pathology of Genomes of University of Nice Sophia-Antipolis, CNRS UMR6267/INSERM U998, Faculty of Medicine, Nice, France; 4Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Università di Pavia, Pavia, Italy; 5Department of Medical Genetics, CHU of Nice, Nice, France The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mech- anisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian te- lomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind.