UNIT-I DNA: Definition, Structure & Discovery DNA
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Sequence-Selective Recognition of Double-Stranded RNA And
Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Hnedzko et al. Sequence-Selective Recognition of Double-Stranded RNA and Enhanced Cellular Uptake of Cationic Nucleobase and Backbone- Modified Peptide Nucleic Acids Dziyana Hnedzko1,*, Dennis W. McGee,2 Yannis A. Karamitas1 and Eriks Rozners1,* Departments of 1 Chemistry and 2 Biological Sciences, Binghamton University, The State University of New York, Binghamton, New York 13902, United States. * To whom correspondence should be addressed. Tel: +1-607-777-2441; Fax: +1-607-777- 4478; Email: [email protected] and [email protected] Running Tittle: RNA recognition and cellular uptake of PNA 1 Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Hnedzko et al. ABSTRACT Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research and biotechnology. However, specific recognition of structured RNA is challenging and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (Ka = 9.4 to 17 × 107 M-1) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA-peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same 7 -1 sequence (Ka = 0.05 to 0.11 × 10 M ). The binding of double-stranded RNA by M-modified 4 -1 -1 PNA-peptide conjugates was a relatively fast process (kon = 2.9 × 10 M s ) compared to 3 -1 -1 the notoriously slow triple helix formation by oligodeoxynucleotides (kon ~ 10 M s ). -
Chapter 14: Functional Genomics Learning Objectives
Chapter 14: Functional Genomics Learning objectives Upon reading this chapter, you should be able to: ■ define functional genomics; ■ describe the key features of eight model organisms; ■ explain techniques of forward and reverse genetics; ■ discuss the relation between the central dogma and functional genomics; and ■ describe proteomics-based approaches to functional genomics. Outline : Functional genomics Introduction Relation between genotype and phenotype Eight model organisms E. coli; yeast; Arabidopsis; C. elegans; Drosophila; zebrafish; mouse; human Functional genomics using reverse and forward genetics Reverse genetics: mouse knockouts; yeast; gene trapping; insertional mutatgenesis; gene silencing Forward genetics: chemical mutagenesis Functional genomics and the central dogma Approaches to function; Functional genomics and DNA; …and RNA; …and protein Proteomic approaches to functional genomics CASP; protein-protein interactions; protein networks Perspective Albert Blakeslee (1874–1954) studied the effect of altered chromosome numbers on the phenotype of the jimson-weed Datura stramonium, a flowering plant. Introduction: Functional genomics Functional genomics is the genome-wide study of the function of DNA (including both genes and non-genic regions), as well as RNA and proteins encoded by DNA. The term “functional genomics” may apply to • the genome, transcriptome, or proteome • the use of high-throughput screens • the perturbation of gene function • the complex relationship of genotype and phenotype Functional genomics approaches to high throughput analyses Relationship between genotype and phenotype The genotype of an individual consists of the DNA that comprises the organism. The phenotype is the outward manifestation in terms of properties such as size, shape, movement, and physiology. We can consider the phenotype of a cell (e.g., a precursor cell may develop into a brain cell or liver cell) or the phenotype of an organism (e.g., a person may have a disease phenotype such as sickle‐cell anemia). -
From Genes to Genomes
From Genes to Genomes Third Edition Concepts and Applications of DNA Technology Jeremy W. Dale, Malcolm von Schantz and Nick Plant University of Surrey, UK ©WILEY-BLACKWELL A John Wiley & Sons, Ltd., Publication Preface xiii 1 From Genes to Genomes 1 1.1 Introduction 1 1.2 Bask molecular biology 4 1.2.1 The DNA backbone 4 1.2.2 The base pairs 6 1.2.3 RNA structure 10 1.2.4 Nucleic acid synthesis 11 1.2.5 Coiling and supercoilin 11 1.3 What is a gene? 13 1.4 Information flow: gene expression 15 1.4.1 Transcription 16 1.4.2 Translation 19 1.5 Gene structure and organisation 20 1.5.1 Operons 20 1.5.2 Exons and introns 21 1.6 Refinements of the model 22 2 How to Clone a Gene 25 2.1 What is cloning? 25 2.2 Overview of the procedures 26 2.3 Extraction and purification of nucleic acids 29 2.3.1 Breaking up cells and tissues 29 2.3.2 Alkaline denaturation 31 2.3.3 Column purification 31 2.4 Detection and quantitation of nucleic acids 32 2.5 Gel electrophoresis 33 2.5.1 Analytical gel electrophoresis 33 2.5.2 Preparative gel electrophoresis 36 vi CONTENTS 2.6 Restriction endonudeases 36 2.6.1 Specificity 37 2.6.2 Sticky and blunt ends 40 2.7 Ligation 42 2.7.1 Optimising Ligation conditions 44 2.7.2 Preventing unwanted Ligation: alkaline phosphatase and double digests 46 2.7.3 Other ways of joining DNA fragments 48 2.8 Modification of restriction fragment ends 49 2.8.1 Linkers and adaptors 50 2.8.2 Homopolymer tailing 52 2.9 Plasmid vectors 53 2.9.1 Plasmid replication 54 2.9.2 Cloning sites 55 2.9.3 Selectable markers 57 2.9.4 Insertional inactivation -
Thermodynamics of DNA Binding and Break Repair by the Pol I DNA
Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus Yanling Yang Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Yang, Yanling, "Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus" (2011). LSU Doctoral Dissertations. 3092. https://digitalcommons.lsu.edu/gradschool_dissertations/3092 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THERMODYNAMICS OF DNA BINDING AND BREAK REPAIR BY THE POL I DNA POLYMERASES FROM ESCHERICHIA COLI AND THERMUS AQUATICUS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Yanling Yang B.S., Qufu Normal University, 2001 M.S., Institute of Microbiology, Chinese Academy Sciences, 2004 M.S. in statistics, Louisiana State University, 2009 December 2011 ACKNOWLEDGMENTS I would like to express my sincere gratitude to some of the people without whose support and guidance none of this work would have been possible. I deeply thank my advisor, Dr. Vince J. LiCata. His guidance and suggestions have been critical to the completion of this research work and the writing of this dissertation. -
Rheumatoid Arthritis a Virus Disease?
J Clin Pathol: first published as 10.1136/jcp.s3-12.1.132 on 1 January 1978. Downloaded from J. clin. Path., 31, Suppl. (Roy. Coll. Path.), 12, 132-143 Inflammation and fibrosis Rheumatoid arthritis a virus disease? A. M. DENMAN From the Division ofImmunological Medicine, Clinical Research Centre, Harrow, Middlesex We now understand many of the immunopatho- arteritis with perivascular infiltrates of chronic logical processes that damage joints and other inflammatory cells, and aberrant immune responses. structures in rheumatoid arthritis and diffuse con- Secondly, virus-infected cells initiate the whole nective tissue diseases. Unfortunately, our progress spectrum of inflammatory and immune reactions in this direction is matched by an equal failure to which characterise the human disorders. These identify the causes of all but a few of these disorders. include complement activation by the classical and Nevertheless, the stimuli which initiate these diseases alternative pathways, which in turn initiate such are probably commonly encountered and tissue processes as the immediate hypersensitivity reaction, damage results when these immunopathological platelet aggregation, and the chemotaxis of granulo- processes are abnormal in intensity and duration- cytes and mononuclear phagocytes. Furthermore, in other words, disease follows an unusual host virus-infected cells attract antibody either through reaction to a variety of environmental factors. This virus-coded antigens on the cell membrane or principle is well illustrated by the degenerative because infected cells commonly carry receptors for copyright. disease of the central nervous system, subacute the Fc portion of the immunoglobulin molecule. sclerosing panencephalitis, which ensues in in- These cells also attract a cellular response in the dividuals who are unable to control the growth form of both specifically sensitised T lymphocytes and dissemination of measles virus. -
Nuclear and Mitochondrial DNA Sequences from Two Denisovan Individuals
Nuclear and mitochondrial DNA sequences from two Denisovan individuals Susanna Sawyera,1, Gabriel Renauda,1, Bence Violab,c,d, Jean-Jacques Hublinc, Marie-Theres Gansaugea, Michael V. Shunkovd,e, Anatoly P. Dereviankod,f, Kay Prüfera, Janet Kelsoa, and Svante Pääboa,2 aDepartment of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany; bDepartment of Anthropology, University of Toronto, Toronto, ON M5S 2S2, Canada; cDepartment of Human Evolution, Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany; dInstitute of Archaeology and Ethnography, Russian Academy of Sciences, Novosibirsk, RU-630090, Russia; eNovosibirsk National Research State University, Novosibirsk, RU-630090, Russia; and fAltai State University, Barnaul, RU-656049, Russia Contributed by Svante Pääbo, October 13, 2015 (sent for review April 16, 2015; reviewed by Hendrik N. Poinar, Fred H. Smith, and Chris B. Stringer) Denisovans, a sister group of Neandertals, have been described on DNA to the ancestors of present-day populations across Asia the basis of a nuclear genome sequence from a finger phalanx and Oceania suggests that in addition to the Altai Mountains, (Denisova 3) found in Denisova Cave in the Altai Mountains. The they may have lived in other parts of Asia. In addition to the only other Denisovan specimen described to date is a molar (Deni- finger phalanx, a molar (Denisova 4) was found in the cave in sova 4) found at the same site. This tooth carries a mtDNA se- 2000. Although less than 0.2% of the DNA in the tooth derives quence similar to that of Denisova 3. Here we present nuclear from a hominin source, the mtDNA was sequenced and differed DNA sequences from Denisova 4 and a morphological description, from the finger phalanx mtDNA at only two positions, suggesting as well as mitochondrial and nuclear DNA sequence data, from it too may be from a Denisovan (2, 3). -
Barriers to Adoption of GM Crops
Iowa State University Capstones, Theses and Creative Components Dissertations Fall 2021 Barriers to Adoption of GM Crops Madeline Esquivel Follow this and additional works at: https://lib.dr.iastate.edu/creativecomponents Part of the Agricultural Education Commons Recommended Citation Esquivel, Madeline, "Barriers to Adoption of GM Crops" (2021). Creative Components. 731. https://lib.dr.iastate.edu/creativecomponents/731 This Creative Component is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Creative Components by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Barriers to Adoption of GM Crops By Madeline M. Esquivel A Creative Component submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Plant Breeding Program of Study Committee: Walter Suza, Major Professor Thomas Lübberstedt Iowa State University Ames, Iowa 2021 1 Contents 1. Introduction ................................................................................................................................. 3 2. What is a Genetically Modified Organism?................................................................................ 9 2.1 The Development of Modern Varieties and Genetically Modified Crops .......................... 10 2.2 GM vs Traditional Breeding: How Are GM Crops Produced? -
Cloning of Gene Coding Glyceraldehyde-3-Phosphate Dehydrogenase Using Puc18 Vector
Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library European Journal of Experimental Biology, 2015, 5(3):52-57 ISSN: 2248 –9215 CODEN (USA): EJEBAU Cloning of gene coding glyceraldehyde-3-phosphate dehydrogenase using puc18 vector Manoj Kumar Dooda, Akhilesh Kushwaha *, Aquib Hasan and Manish Kushwaha Institute of Transgene Life Sciences, Lucknow (U.P), India _____________________________________________________________________________________________ ABSTRACT The term recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refers to the same process. Gene cloning is a set of experimental methods in molecular biology and useful in many areas of research and for biomedical applications. It is the production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques. cDNA is synthesized by using template RNA isolated from blood sample (human). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Amplify the gene (GAPDH) using primer forward and reverse with the sequence of 5’-TGATGACATCAAGAAGGTGGTGAA-3’ and 5’-TCCTTGGAGGCCATGTGGGCCAT- 3’.pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique and cleaved with the help of SmaI restriction enzyme. Modern cloning vectors include selectable markers (most frequently antibiotic resistant marker) that allow only cells in which the vector but necessarily the insert has been transfected to grow. Additionally the cloning vectors may contain color selection markers which provide blue/white screening (i.e. alpha complementation) on X- Gal and IPTG containing medium. Keywords: RNA isolation; TRIzol method; Gene cloning; Blue/white screening; Agarose gel electrophoresis. -
Molecular Basis of the Function of Transcriptional Enhancers
cells Review Molecular Basis of the Function of Transcriptional Enhancers 1,2, 1, 1,3, Airat N. Ibragimov y, Oleg V. Bylino y and Yulii V. Shidlovskii * 1 Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia; [email protected] (A.N.I.); [email protected] (O.V.B.) 2 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia 3 I.M. Sechenov First Moscow State Medical University, 8, bldg. 2 Trubetskaya St., 119048 Moscow, Russia * Correspondence: [email protected]; Tel.: +7-4991354096 These authors contributed equally to this study. y Received: 30 May 2020; Accepted: 3 July 2020; Published: 5 July 2020 Abstract: Transcriptional enhancers are major genomic elements that control gene activity in eukaryotes. Recent studies provided deeper insight into the temporal and spatial organization of transcription in the nucleus, the role of non-coding RNAs in the process, and the epigenetic control of gene expression. Thus, multiple molecular details of enhancer functioning were revealed. Here, we describe the recent data and models of molecular organization of enhancer-driven transcription. Keywords: enhancer; promoter; chromatin; transcriptional bursting; transcription factories; enhancer RNA; epigenetic marks 1. Introduction Gene transcription is precisely organized in time and space. The process requires the participation of hundreds of molecules, which form an extensive interaction network. Substantial progress was achieved recently in our understanding of the molecular processes that take place in the cell nucleus (e.g., see [1–9]). -
Hammerhead Ribozymes Against Virus and Viroid Rnas
Hammerhead Ribozymes Against Virus and Viroid RNAs Alberto Carbonell, Ricardo Flores, and Selma Gago Contents 1 A Historical Overview: Hammerhead Ribozymes in Their Natural Context ................................................................... 412 2 Manipulating Cis-Acting Hammerheads to Act in Trans ................................. 414 3 A Critical Issue: Colocalization of Ribozyme and Substrate . .. .. ... .. .. .. .. .. ... .. .. .. .. 416 4 An Unanticipated Participant: Interactions Between Peripheral Loops of Natural Hammerheads Greatly Increase Their Self-Cleavage Activity ........................... 417 5 A New Generation of Trans-Acting Hammerheads Operating In Vitro and In Vivo at Physiological Concentrations of Magnesium . ...... 419 6 Trans-Cleavage In Vitro of Short RNA Substrates by Discontinuous and Extended Hammerheads ........................................... 420 7 Trans-Cleavage In Vitro of a Highly Structured RNA by Discontinuous and Extended Hammerheads ........................................... 421 8 Trans-Cleavage In Vivo of a Viroid RNA by an Extended PLMVd-Derived Hammerhead ........................................... 422 9 Concluding Remarks and Outlooks ........................................................ 424 References ....................................................................................... 425 Abstract The hammerhead ribozyme, a small catalytic motif that promotes self- cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically -
Molecular Biology and Applied Genetics
MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included. -
Ribozymes Targeted to the Mitochondria Using the 5S Ribosomal Rna
RIBOZYMES TARGETED TO THE MITOCHONDRIA USING THE 5S RIBOSOMAL RNA By JENNIFER ANN BONGORNO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Jennifer Bongorno To my grandmother, Hazel Traster Miller, whose interest in genealogy sparked my interest in genetics, and without whose mitochondria I would not be here ACKNOWLEDGMENTS I would like to thank all the members of the Lewin lab; especially my mentor, Al Lewin. Al was always there for me with suggestions and keeping me motivated. He and the other members of the lab were like my second family; I would not have had an enjoyable experience without them. Diana Levinson and Elizabeth Bongorno worked with me on the fourth and third mouse transfections respectively. Joe Hartwich and Al Lewin tested some of the ribozymes in vitro and cloned some of the constructs I used. James Thomas also helped with cloning and was an invaluable lab manager. Verline Justilien worked on a related project and was a productive person with whom to bounce ideas back and forth. Lourdes Andino taught me how to use the new phosphorimager for my SYBR Green-stained gels. Alan White was there through it all, like the older brother I never had. Mary Ann Checkley was with me even longer than Alan, since we both came to Florida from Ohio Wesleyan, although she did manage to graduate before me. Jia Liu and Frederic Manfredsson were there when I needed a beer.