Internal Control for Real-Time Polymerase Chain Reaction Based on MS2 Bacteriophage for RNA Viruses Diagnostics
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Asymmetric Cryo-EM Reconstruction of Phage MS2 Reveals Genome Structure in Situ
ARTICLE Received 8 Feb 2016 | Accepted 11 Jul 2016 | Published 26 Aug 2016 DOI: 10.1038/ncomms12524 OPEN Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ Roman I. Koning1,2, Josue Gomez-Blanco3, Inara Akopjana4, Javier Vargas3, Andris Kazaks4, Kaspars Tars4, Jose´ Marı´a Carazo3 & Abraham J. Koster1,2 In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly. 1 Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. 2 Netherlands Centre for Electron Nanoscopy, Institute of Biology Leiden, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. 3 Biocomputing Unit, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CNB-CSIC), Darwin 3, Campus Universidad Auto´noma, Cantoblanco, Madrid 28049, Spain. 4 Biomedical Research and Study Centre, Ratsupites 1, LV-1067 Riga, Latvia. Correspondence and requests for materials should be addressed to R.I.K. (email: [email protected]). -
Encapsulation of Biomolecules in Bacteriophage MS2 Viral Capsids
Encapsulation of Biomolecules in Bacteriophage MS2 Viral Capsids By Jeff Edward Glasgow A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in Charge: Professor Matthew Francis, Co-Chair Professor Danielle Tullman-Ercek, Co-Chair Professor Michelle Chang Professor John Dueber Spring 2014 Encapsulation of Biomolecules in Bacteriophage MS2 Viral Capsids Copyright ©2014 By: Jeff Edward Glasgow Abstract Encapsulation of Biomolecules in Bacteriophage MS2 Viral Capsids By Jeff Edward Glasgow Doctor of Philosophy in Chemistry University of California, Berkeley Professor Matthew Francis, Co-Chair Professor Danielle Tullman-Ercek, Co-Chair Nanometer-scale molecular assemblies have numerous applications in materials, catalysis, and medicine. Self-assembly has been used to create many structures, but approaches can match the extraordinary combination of stability, homogeneity, and chemical flexibility found in viral capsids. In particular, the bacteriophage MS2 capsid has provided a porous scaffold for several engineered nanomaterials for drug delivery, targeted cellular imaging, and photodynamic thera- py by chemical modification of the inner and outer surfaces of the shell. This work describes the development of new methods for reassembly of the capsid with concomitant encapsulation of large biomolecules. These methods were then used to encapsulate a variety of interesting cargoes, including RNA, DNA, protein-nucleic acid and protein-polymer conjugates, metal nanoparticles, and enzymes. To develop a stable, scalable method for encapsulation of biomolecules, the assembly of the capsid from its constituent subunits was analyzed in detail. It was found that combinations of negatively charged biomolecules and protein stabilizing agents could enhance reassembly, while electrostatic interactions of the biomolecules with the positively charged inner surface led to en- capsulation. -
RNA-Dependent Amplification of Mammalian Mrna Encoding Extracellular Matrix Components
bioRxiv preprint doi: https://doi.org/10.1101/376293; this version posted December 5, 2018. 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. RNA-DEPENDENT AMPLIFICATION OF MAMMALIAN mRNA ENCODING EXTRACELLULAR MATRIX COMPONENTS: IDENTIFICATION OF CHIMERIC RNA INTERMEDIATES FOR a1, b1, AND g1 CHAINS OF LAMININ. * Vladimir Volloch 1 , Sophia Rits 2,3, Bjorn Olsen 1 1: Department of Developmental Biology, Harvard School of Dental Medicine 2: Howard Hughes Medical Institute at Children’s Hospital, Boston 3: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School *: Corresponding Author, [email protected]; [email protected] ABSTRACT. The aim of the present study was to test for the occurrence of key elements predicted by the previously postulated mammalian RNA-dependent mRNA amplification model in a tissue producing massive amounts of extracellular matrix proteins. At the core of RNA-dependent mRNA amplification, until now only described in one mammalian system, is the self-priming of an antisense strand and extension of its 3’ terminus into a sense-oriented RNA containing the protein-coding information of a conventional mRNA. The resulting product constitutes a new type of biomolecule. It is chimeric in that it contains covalently connected antisense and sense sequences in a hairpin configuration. Cleavage of this chimeric intermediate in the loop region of a hairpin structure releases mRNA which contains an antisense segment in its 5’UTR; depending on the position of self-priming, the chimeric end product may encode the entire protein or its C-terminal fragment. -
Sensitive Detection of Live Escherichia Coli by Bacteriophage Amplification-Coupled
bioRxiv preprint doi: https://doi.org/10.1101/318071; this version posted May 9, 2018. 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. 1 Sensitive detection of Live Escherichia coli by bacteriophage amplification-coupled 2 immunoassay on the Luminex® MAGPIX instrument 3 Tomotaka Midoa*, Eric M. Schafferb,c, Robert W. Dorseyc, Shanmuga Sozhamannand,e, E. 4 Randal Hofmannc,f 5 6 CBRN Detection Technology Section, CBRN Defense Technology Division, Advanced Defense 7 Technology Center, Acquisition, Technology and Logistics Agency (ATLA), Tokyo, Japana; 8 Leidos, Inc., Aberdeen Proving Ground, Maryland, USAb; Biosciences Division, U.S. Army 9 Edgewood Chemical Biological Center, Aberdeen Proving Grounds, Edgewood, MD, USAc; The 10 Tauri Group, LLC, Alexandria, VA, USAd; Defense Biological Product Assurance Office, JPM 11 G, JPEO, Frederick, MD, USAe; EXCET, Inc. Springfield, VA, USAf. 12 13 Running Head: Bacterial detection via phage on a MAGPIX instrument 14 15 #Address correspondence to [email protected] 16 *Present address: International Affairs Office, International Cooperation Division, Department 17 of Equipment Policy, Acquisition, Technology and Logistics Agency (ATLA), Tokyo, Japan 18 19 20 1 bioRxiv preprint doi: https://doi.org/10.1101/318071; this version posted May 9, 2018. 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. 21 Abstract (250 words) 22 Phages are natural predators of bacteria and have been exploited in bacterial detection because of 23 their exquisite specificity to their cognate bacterial hosts. -
RNA-Guided Transcriptional Regulation in Plants Via Dcas9 Chimeric Proteins Thesis by Hatoon Baazim in Partial Fulfillment of Th
RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins Thesis by Hatoon Baazim In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia May 2014 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Hatoon Baazim is approved by the examination committee. Committee Chairperson: Magdy Mahfouz Committee Member: Christoph Gehring Committee Member: Samir Hamdan 3 © Approval Date May 2014 Hatoon Baazim All Rights Reserved 4 ABSTRACT RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins Hatoon Baazim Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. -
The PVT Gene Frequently Amplifies with MYC in Tumor Cells E
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1989, p. 1148-1154 Vol. 9, No. 3 0270-7306/89/031148-07$02.OO/O Copyright ©3 1989, American Society for Microbiology The PVT Gene Frequently Amplifies with MYC in Tumor Cells E. SHTIVELMAN AND J. MICHAEL BISHOP* Department of Microbiology and Immunology and The G. W. Hooper Research Foundation, University of California Medical Center, San Francisco, California 94143 Received 5 October 1988/Accepted 8 December 1988 The line of human colon carcinoma cells known as COL0320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 ofMYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion ofPVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification ofMYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC. -
What Is a Gene, Post-ENCODE? History and Updated Definition
Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Perspective What is a gene, post-ENCODE? History and updated definition Mark B. Gerstein,1,2,3,9 Can Bruce,2,4 Joel S. Rozowsky,2 Deyou Zheng,2 Jiang Du,3 Jan O. Korbel,2,5 Olof Emanuelsson,6 Zhengdong D. Zhang,2 Sherman Weissman,7 and Michael Snyder2,8 1Program in Computational Biology & Bioinformatics, Yale University, New Haven, Connecticut 06511, USA; 2Molecular Biophysics & Biochemistry Department, Yale University, New Haven, Connecticut 06511, USA; 3Computer Science Department, Yale University, New Haven, Connecticut 06511, USA; 4Center for Medical Informatics, Yale University, New Haven, Connecticut 06511, USA; 5European Molecular Biology Laboratory, 69117 Heidelberg, Germany; 6Stockholm Bioinformatics Center, Albanova University Center, Stockholm University, SE-10691 Stockholm, Sweden; 7Genetics Department, Yale University, New Haven, Connecticut 06511, USA; 8Molecular, Cellular, & Developmental Biology Department, Yale University, New Haven, Connecticut 06511, USA While sequencing of the human genome surprised us with how many protein-coding genes there are, it did not fundamentally change our perspective on what a gene is. In contrast, the complex patterns of dispersed regulation and pervasive transcription uncovered by the ENCODE project, together with non-genic conservation and the abundance of noncoding RNA genes, have challenged the notion of the gene. To illustrate this, we review the evolution of operational definitions of a gene over the past century—from the abstract elements of heredity of Mendel and Morgan to the present-day ORFs enumerated in the sequence databanks. We then summarize the current ENCODE findings and provide a computational metaphor for the complexity. -
The Effects of L Protein on Mreb and Cell Lysis
The Effects of L protein on MreB and Cell Lysis Authors: Alannah Templon, Reddy Karthik, Ry Young, Randy Morgenstein * Abstract: The MS2 bacteriophage is a single-stranded, positive-strand RNA virus that contains four genes: mat, coat, rep, and L. The L protein is responsible for cell lysis, although relatively little is known about its mode of action. Unlike other viral lysis proteins, which inhibit cell wall synthesis at the division site causing midcell blebs, L protein appears to cause lysis at random cellular locations, as seen by bleb formation throughout the cell. We hypothesized that L protein works with MreB, an essential protein for cell wall synthesis, which is localized throughout the cell body. We seek to identify how L protein affects MreB by possibly: causing the mislocalization of MreB at specific sites of lysis, activating a section of MreB to form a hotspot for cell wall synthesis, or deactivating a section of the MreB pool, causing cell defects. To begin to determine which mechanism is correct, we will measure the localization of MreB in cells undergoing lysis to see if there is a correlation between MreB localization and L protein induced lysis. We will then examine if there is a direct interaction between L protein and MreB using biomolecular fluorescent complementation. Learning how L protein lyses cells will provide us with a better understanding of single gene lysis, which can be applied to phage therapy to kill disease causing bacteria and to effectively prevent bacteriophages from killing helpful bacteria that assist in preventing disease or are important for industrial purposes. -
Bacterial Retrons Function in Anti-Phage Defense
Article Bacterial Retrons Function In Anti-Phage Defense Graphical Abstract Authors Adi Millman, Aude Bernheim, Retrons appear in an operon Retrons generate an RNA-DNA hybrid via reverse transcription with additional “effector” genes Avigail Stokar-Avihail, ..., Azita Leavitt, ncRNA Reverse Transcriptase (RT) msDNA Ribosyltransferase Yaara Oppenheimer-Shaanan, (RNA-DNA hybrid) DNA-binding RT Rotem Sorek RNA Retron function 2 transmembrane 5’ G RT domains 2’-5’ 3’ was unknown 3’ RT Correspondence cDNA RT Cold-shock [email protected] 5’ G 2’ 3’ G G cDNA RT RT ATPase Nuclease In Brief reverse transcription RNase H Retrons are part of a large family of anti- Retrons protect bacteria from phage Inhibition of RecBCD by phages triggers retron Ec48 defense phage defense systems that are widespread in bacteria and confer Bacterial density during phage infection Growth resistance against a broad range of B Effector arrest RT D RT activation with retron C B phages, mediated by abortive infection. Effector no retron Ec48 “guards” the bacterial Effector activation leads RecBCD complex to abortive infection bacterial density Retron Ec48 RT 2TM time RecBCD inhibitor B B D D RT Bacteria without retron Bacteria with retron C C B B Phage proteins inhibit RecBCD Retron Ec48 senses RecBCD inhibition Highlights d Retrons are preferentially located in defense islands d Retrons, together with their effector genes, protect bacteria from phages d Protection from phage is mediated by abortive infection d Retron Ec48 guards RecBCD. Inhibition of RecBCD by phages triggers retron defense Millman et al., 2020, Cell 183, 1–11 December 10, 2020 ª 2020 Elsevier Inc. -
Review of Coliphages As Possible Indicators of Fecal Contamination for Ambient Water Quality
REVIEW OF COLIPHAGES AS POSSIBLE INDICATORS OF FECAL CONTAMINATION FOR AMBIENT WATER QUALITY 820-R-15-098 EPA Office of Water Office of Science and Technology Health and Ecological Criteria Division April 17, 2015 NOTICES This document has been drafted and approved for publication by the Health and Ecological Criteria Division, Office of Science and Technology, United States (U.S.) Environmental Protection Agency (EPA), and is approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. i ACKNOWLEDGMENTS The development of this criteria document was made possible through an effort led by Sharon Nappier, EPA Project Manager, EPA, Office of Science and Technology, Office of Water. EPA acknowledges the valuable contributions of EPA Internal Technical Reviewers who reviewed this document: Jamie Strong and Elizabeth Doyle. The project described here was managed by the Office of Science and Technology, Office of Water, EPA under EPA Contract EP-C-11-005 to ICF International. EPA also wishes to thank Audrey Ichida, Kirsten Jaglo, Jeffrey Soller, Arun Varghese, Alexandria Boehm, Kara Nelson, Margaret Nellor, and Kaedra Jones for their contributions and invaluable support. The primary contact regarding questions or comments to this document is: Sharon Nappier U.S. EPA Headquarters Office of Science and Technology, Office of Water 1200 Pennsylvania Ave., NW Washington, DC 20460 Mail Code 4304T Phone: (202) 566-0740 Email: [email protected] ii EXTERNAL PEER REVIEW WORKGROUP The External Peer Review was managed by the Office of Science and Technology, Office of Water, EPA under EPA Contract No. EP-C-13-010 to Versar, Inc. -
RNA-Dependent Synthesis of Mammalian Mrna: Identification of Chimeric Intermediate and Putative End Product
bioRxiv preprint doi: https://doi.org/10.1101/071266; this version posted August 25, 2016. 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. RNA-DEPENDENT SYNTHESIS OF MAMMALIAN mRNA: IDENTIFICATION OF CHIMERIC INTERMEDIATE AND PUTATIVE END PRODUCT # Sophia Rits 1,2, Bjorn R. Olsen3, Vladimir Volloch3, 1: Howard Hughes Medical Institute at Children’s Hospital, Boston USA 2: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School 3: Dept. of Developmental Biology, Harvard School of Dental Medicine # : Corresponding author, [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/071266; this version posted August 25, 2016. 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. ABSTRACT. Our initial understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the “central dogma of molecular biology”, was eventually amended to account for the information back-flow from RNA to DNA (reverse transcription), and for its “side-flow” from RNA to RNA (RNA-dependent RNA synthesis, RdRs). These processes, both potentially leading to protein production, were described only in viral systems, and although putative RNA-dependent RNA polymerase (RdRp) was shown to be present, and RdRs to occur, in most, if not all, mammalian cells, its function was presumed to be restricted to regulatory. Here we report the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. -
Crystal Structure of an Okazaki Fragment at 2-A Resolution
Proc. Nadi. Acad. Sci. USA Vol. 89, pp. 534-538, January 1992 Biochemistry Crystal structure of an Okazaki fragment at 2-A resolution (RNA-DNA hybrid/replication/x-ray crystalbography/shock-freezing/A-DNA to B-DNA transition) MARTIN EGLI, NASSIM USMAN, SHUGUANG ZHANG, AND ALEXANDER RICH Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Contributed by Alexander Rich, October 17, 1991 ABSTRACT Ini DNA replication, Okazaki fragments are 10 9 8 7 6 5 4 3 2 1 formed as double-stranded intermediates during synthesis of 3' C G C A T A T G G G 5' the lagging strand. They are composed of the growing DNA 5' G C T 3, strand primed by RNA and the template strand. The DNA GI A T A C C C oligonucleotide d(GGGTATACGC) and the chimeric RNA- 1 1 12 13 14 15 16 17 18 19 20 DNA oligonucleotide r(GCG)d(TATACCC) were combined to FIG. 1. Sequence of the Okazaki fragment (RNA residues form a synthetic Okazaki fragment and its three-dimensional Note twofold rotational symmetry. structure was determined by x-ray crystallography. The frag- boxed). the lack of ment adopts an overall A-type conformation with 11 residues were non-self-complementary to avoid pairing of each of the per turn. Although the base-pair geometry, particularly in the strands with itself (Fig. 1). The DNA decamer was prepared central TATA part, is distorted, there is no evidence for a conventionally by the automated, phosphoramidite method transition from the A- to the B-type conformation at the on an Applied Biosystems model 380B DNA synthesizer.