DOCSLIB.ORG

Expression of Artemia LEA Proteins in Drosophila Melanogaster Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Expression of Artemia LEA Proteins in Drosophila Melanogaster Cells Eastern Illinois University The Keep Masters Theses Student Theses & Publications 2016 Expression of Artemia LEA Proteins in Drosophila melanogaster Cells Using Multicistronic Vector Constructs Kazi Nazrul Islam Eastern Illinois University This research is a product of the graduate program in Biological Sciences at Eastern Illinois University. Find out more about the program. Recommended Citation Islam, Kazi Nazrul, "Expression of Artemia LEA Proteins in Drosophila melanogaster Cells Using Multicistronic Vector Constructs" (2016). Masters Theses. 2454. https://thekeep.eiu.edu/theses/2454 This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. The Graduate School� EAsTillt"llLLINOIS UN!VERSlTY­ Thesis Maintenance and Reproduction Certificate FOR: Graduate Candidates Completing Theses in Partial Fulfillment of the Degree Graduate Faculty Advisors Directing the Theses RE: Preservation, Reproduction, and Distribution of Thesis Research Preserving, reproducing, and distributing thesis research is an important part of Booth Library's responsibility to provide access to scholarship. In order to further this goal, Booth Library makes all graduate theses completed as part of a degree program at Eastern Illinois University available for personal study, research, and other not-for-profit educational purposes. Under 17 U.S.C. § 108, the library may reproduce and distribute a copy without infringing on copyright; however, professional courtesy dictates that permission be requested from the author before doing so. Your signatures affirm the following: • The graduate candidate is the author of this thesis. • The graduate candidate retains the copyright and intellectual property rights associated with the original research, creative activity, and intellectual or artistic content of the thesis. • The graduate candidate certifies her/his compliance with federal copyright law (Title 17 of the U. S. Code) and her/his right to authorize reproduction and distribution of all copyrighted materials included in this thesis. • The graduate candidate in consultation with the faculty advisor grants Booth Library the non­ exclusive, perpetual right to make copies of the thesis freely and publicly available without restriction, by means of any current or successive technology, including by not limited to photocopying, microfilm, digitization, or internet. • The graduate candidate acknowledges that by depositing her/his thesis with Booth Library, her/his work is available for viewing by the public and may be borrowed through the library's circulation and interlibrary loan departments, or accessed electronically. • The graduate candidate waives the confidentiality provisions of the Family Educational Rights and Privacy Act (FERPA) (20 U. S. C. § 1232g; 34 CFR Part 99) with respect to the contents of the thesis and with respect to information concerning authorship of the thesis, including name and status as a student at Eastern Illinois University. I have conferred with my graduate faculty advisor. My signature below indicates that I have read and agree with the above statements, and hereby give my permission to allow Booth Library to reproduce and distribute my thesis. My adviser's signature indicates concurrence to reproduce and distribute the thesis. / Graduate Candidate Signature F?culty Adviser Signature Kazi Nazrul Islam Michael A. Menze Printed Name Printed Name Biological Sciences Ul ( l1,/) L�b Graduate Degree Program Date Please submit in duplicate. Expression of Artemia LEA proteins in Drosophila melanogaster cells using multicistronic vector constructs (TITLE) BY Kazi Nazrul Islam THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in Biological Sciences IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2016 YEAR I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE 07' )''>{ ) ('\ i. 1/lf/i 7 . THESIS COMMITTEE CHAIR DATE Ut::l"'AKlcTc I /:::>IJHUUL IJHAIK DATE CHAIR'S DESIGNEE 1 �¢6 . " ruc�I� rl"a(a..U.Alricc lAClllDCC DATE THESIS COMMITTEE MEMBER DATE _]j_2-{pIi(., THESIS COMMlhEE MEMBER DATE THESIS COMMITTEE MEMBER DATE TABLE OF CONTENTS PAGE NUMBER ABSTRACT 3 ACKNOWLEDGEMENTS 4 LIST OF TABLES AND FIGURES 5 INTRODUCTION 9 MATERIALS AND METHODS 23 RESULTS 39 DISCUSSION 65 CONCLUSION 72 REFERENCES 73 2 ABSTRACT In nature several organisms exhibit anhydrobiosis, the outstanding featureto survive in extreme desiccation by entering into a state of dormancy known as diapause. The cyst of the brine shrimp Artemia franciscana shows anhydrobiosis by entering into a diapause phase. Previous studies showed a correlation between anhydrobiosis and expression of highly hydrophilic polypeptides termed late embryogenesis abundant (LEA) proteins. However, the precise molecular mechanisms of LEA proteins are still unknown. The presence of multiple LEA proteins in Artemia suggests that some of them might work together. Here, I aimed to express different combination of two LEA proteins fromArtemia .franciscana in the Drosophila melanogaster cell lines Kcl67 and S2R+ by using the multicistronic vector pAc5-STABLE2-Neo. lmmunoblot confirmed concurrent expression of both mCherry-LEA3m and GFP-LEA6 proteins in the Kc 167 cells transfected with LEA3m+LEA6 construct. However, in three other Kc167 clones, although Western blot verified expression of mCherry tagged LEA proteins transcribed at first position of the vector, GFP tagged LEA protein cloned at second position of the vector was not detected. Another assumption that consensus ribosome recognition sequence of Drosophila would improve expression of LEA proteins in Drosophila cells was supported by the images of fluorescence microscopy. The final goal, simultaneous expression of two LEA proteins without the fluorescent reporters,was partially successfulas immunoblot identifiedonly DDK tagged firstLEA proteins but not 6X His tagged second LEA proteins. Nonetheless, our results showed that expression of two LEA proteins concurrently in the Drosophila cells is possible by using the multicistronic vector instead of conventional two vectors system. 3 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor Dr. Michael A. Menze for his continuous support in my graduate study and research, as well as his perseverance and motivation. His guidance has made my research and writing of this thesis possible. Before joining the lab, I did not have much knowledge in molecular biology but his excellent guidance and depth knowledge made me competent in laboratory techniques as well as theoretical knowledge. I could not have imagined having a better advisor for my graduate study. Besides my advisor, I would like to thank Dr. Gary A. Bulla and Dr. Britto P. Nathan as members of my thesis committee. Their encouragement in my current, as well as future graduate studies helped me to finish writing this thesis. Their helpfulness and insightful questions helped me to produce the most viable results. I also thank my fellow labmates including Brett Janis, Clinton Belott, Daniel Webster, Robert Skolik, David Grimm, David Constantinescu, Fazlul Karim, and Shankar Gurung for their assistance and inspiring discussions and all the funwe have had in the last two years. As an international student, it would be difficult to study and concentrate in research without their cheerful presence. 4 LIST OF TABLES AND FIGURES Title Page # Table 1 LEA proteins found in the brine shrimp Artemia franciscana 11 Table 2 The primers used forcl oning of fluorescent marked LEA 25 proteins in the STABLE-2 vector Table 3 The primers used forcl oning of mCherry marked LEAl.3 26 proteins in the STABLE-2 vector with and without ribosome recognition sequence Table 4 The primers used forPCR amplification and cloning of LEA 27 proteins in the STABLE-2 vector with 6X His and DDK tags Table 5 The reagents used to amplifyDNA through PCR 28 Table 6 The reagents used to set up reactions forrestri ction enzyme 29 digestion Table 7 The reagents used to set up ligation reactions 30 Table 8 Optical density of standard BSA samples in Bradfordreagent 35 at 595 nm Figure 1 Schematic representation of the pAc5-STABLE2-Neo vector 23 Figure 2 PCR amplification ofLEA3m DNA (921 bases) using 40 isolated plasmids from bacterial colonies as template Figure 3 Restriction enzyme digestion of the plasmids that yielded 40 LEA3m DNA in PCR Figure 4 PCR products of LEA6 (771 bases) using isolated plasmids as 41 template DNA 5 Figure 5 Single digestion of the isolated plasmids that showed positive 42 PCR result forgene LEA6 Figure 6 Amplification ofLEAl (1074 bases) and LEA2 (1092 bases) 43 through PCR using isolated plasmids as template Figure 7 Linearized plasmids that showed positive PCR result for 43 LEAl and LEA2 Figure 8 Single digestion of the plasmids purifiedfrom the bacteria 44 cloned with LEAl.3 +LEA6 Figure 9 PCR products of LEA 1.3 ( 600 bases) using isolated plasmids 45 as template DNA Figure 10 Expression of mCherry (red, column 1) and GFP (green, 46 column 2) tagged LEA proteins in Kc167 cell lines Figure 11 Expression of mCherry (red, column 1) and GFP (green, 47 column 2) labeled LEA proteins in S2R+ cell lines Figure 12 Immunoblotting forid entification of fluorescent tagged LEA 49 proteins in the Drosophila Kc 167 cell lines Figure 13 Immunoblotting
Load more

Recommended publications
  • S. Cerevisiae
    DOCTOR OF PHILOSOPHY Saccharomyces Cerevisiae as a biotechnological tool for ageing research studies on translation and metabolism Stephanie Cartwright 2013 Aston University Some pages of this thesis may have been removed for copyright restrictions. If you have discovered material in AURA which is unlawful e.g. breaches copyright, (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please read our Takedown Policy and contact the service immediately SACCHAROMYCES CEREVISIAE AS A BIOTECHNOLOGICAL TOOL FOR AGEING RESEARCH: STUDIES ON TRANSLATION AND METABOLISM STEPHANIE PATRICIA CARTWRIGHT Doctor of Philosophy ASTON UNIVERSITY July 2013 ©Stephanie Patricia Cartwright, 2013 Stephanie Patricia Cartwright asserts her moral right to be identified as the author of this thesis. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgment. 1 ASTON UNIVERSITY SACCHAROMYCES CEREVISIAE AS A BIOTECHNOLOGICAL TOOL FOR AGEING RESEARCH: STUDIES ON TRANSLATION AND METABOLISM Stephanie Patricia Cartwright PhD 2013 Thesis summary The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products.
    [Show full text]
  • The Reoviridae the VIRUSES
    The Reoviridae THE VIRUSES Series Editors HEINZ FRAENKEL-CONRAT, University of California Berkeley, California ROBERT R. WAGNER, University of Virginia School of Medicine Charlottesville, Virginia THE HERPESVIRUSES, Volumes I, 2, 3, and 4 Edited by Bernard Roizman THE REOVIRIDAE Edited by Wolfgang K. Joklik THE PARVOVIRUSES Edited by Kenneth I. Berns The Reoviridae Edited by WOLFGANG K. JOKLIK Duke University Medical Center Durham, North Carolina Springer Science+Business Media, LLC Library of Congress Cataloging in Publication Data Main entry under title: The Reoviridae. (Viruses) Includes bibliographical references and index. 1. Reoviruses. L Joklik, Wolfgang K. II. Series. QR414.R46 1983 576/.64 83-6276 ISBN 978-1-4899-0582-6 ISBN 978-1-4899-0582-6 ISBN 978-1-4899-0580-2 (eBook) DOI 10.1007/978-1-4899-0580-2 © Springer Science+Business Media New York 1983 Originally published by Plenum Press, New York in 1983 Softcover reprint of the hardcover 1st edition 1983 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Con tribu tors Guido 'Boccardo, Istituto di Fitovirologia Applicata del C.N.R., 10135 To­ rino, Italy Bernard N. Fields, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115; and Depart­ ment of Medicine, Brigham and Women's Hospital, Boston, Massa­ chusetts 02115 R. I. B. Fran'cki, Department of Plant Pathology, Waite Agricultural Re­ search Institute, The University of Adelaide, Adelaide 5064, South Australia Barry M.
    [Show full text]
  • UC Berkeley UC Berkeley Electronic Theses and Dissertations
    UC Berkeley UC Berkeley Electronic Theses and Dissertations Title The Effect of Inflammatory Stimuli on Cryptic Peptide Presentation for Immune Surveillance Permalink https://escholarship.org/uc/item/8qt1349m Author Prasad, Sharanya Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California The Effect of Inflammatory Stimuli on Cryptic Peptide Presentation for Immune Surveillance by Sharanya Prasad A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Nilabh Shastri, Chair Professor Russell Vance Professor Astar Winoto Professor Eva Harris Spring 2014 The Effect of Inflammatory Stimuli on Cryptic Peptide Presentation for Immune Surveillance Copyright 2014 by Sharanya Prasad 1 Abstract The Effect of Inflammatory Stimuli on Cryptic Peptide Presentation for Immune Surveillance by Sharanya Prasad Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Nilabh Shastri, Chair Cytolytic T cells eliminate infected cells by recognizing intracellular peptides presented by MHC class I molecules. The antigenic peptides are derived primarily from newly synthesized proteins including those produced by cryptic translation. Previous studies have shown that in addition to the canonical AUG codon, translation can be initiated at non-AUG codons. Furthermore, translation initiation at non-AUG codons such as CUG is mechanistically dis- tinct from canonical translation initiation as it is resistant to protein synthesis inhibitors that cause global translation shutdown. Here, we show that Toll-like receptor (TLR) sig- naling pathways involved in pathogen recognition enhance presentation of the cryptically translated peptides.
    [Show full text]
  • Translational Regulation in Arabidopsis Thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2010 Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3 Bijoyita Roy [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Molecular Genetics Commons Recommended Citation Roy, Bijoyita, "Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3. " PhD diss., University of Tennessee, 2010. https://trace.tennessee.edu/utk_graddiss/845 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Bijoyita Roy entitled "Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Botany. Albrecht G. von Arnim, Major Professor We have read this dissertation and recommend its acceptance: Barry D. Bruce, Bruce D. McKee, David A. Brian Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Bijoyita Roy entitled “Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3”.
    [Show full text]
  • Catalogs | Rutgers University
    Graduate School-- New Brunswick Catalog 1999--2001 Contents Academic Calendars 2 About the University 3 Graduate Study at the University 4 Other Graduate Study at the University 5 About the Cover: Degree Programs Available 6 The antique map of Africa reflects the most current project Admission 8 of the Rutgers Center for Historical Analysis (RCHA). Tuition and Fees 9 The RCHA brings together internationally distinguished scholars, the university community, and the New Jersey Financial Aid 11 public to engage in research, education, and public service Student Services 14 on historical topics of broad community relevance. The RCHA’s programs are designed to put current social trends Academic Policies and Procedures 21 and issues in historical perspective and to overcome the Degree Requirements 32 fragmentation of knowledge that keeps us from seeing the wider dimensions of problems. Programs, Faculty, and Courses 38 The RCHA’s project for the academic years 1997–1999 Research Centers, Bureaus, and Institutes 200 was “The Black Atlantic: Race, Nation, and Gender.” This project traced the globalization of African culture and the Administration 205 formation of the Black Atlantic since the beginning of the Governance of the University 206 modern slave trade. The forced migration of Africans during the modern slave trade marked the beginning of Divisions of the University 207 the globalization of African culture and the formation Maps 212 of the Black Atlantic. Since then, hundreds of thousands of African people have crossed and recrossed the Atlantic, Index 221 and migrated between Atlantic countries. This movement of Africans and their descendants has continually remade diasporic ethnicities and political cultures by allowing Important Notice: for the exchange of religion, aesthetic traditions, expressive Please note that only the printed version of this catalog culture, and strategies for resistance.
    [Show full text]
  • 202103031643031075.Pdf
    CASE STUDY 179 been developed. An example is GenBank­ or others with similar sequences have sequences. Scroll below the table to (http://www.ncbi.nlm.nih.gov/genbank/), been discovered. Enter this sequence see the aligned sequences from this which is the National Institutes of Health into the “Enter Query Sequence” text search, and then answer the following database of all publicly available sequence box at the top of the page. Near the questions: data. This global resource, with access to bottom of the page, under the “Pro- a. What were the top three matches databases in Europe and Japan, currently gram Selection” category, choose to your query sequence? contains more than 220 billion base pairs “blastn”; then click on the “BLAST” of sequence data! button at the bottom of the page to b. For each alignment, BLAST also Earlier (in the Exploring Genomics run the search. It may take several min- indicates the percent identity and exercises for Chapter 7), you were intro- utes for results to be available because the number of gaps in the match duced to the National Center for Biotech- BLAST is using powerful algorithms between the query and subject nology Information (NCBI) Genes and to scroll through billions of bases of sequences. What was the percent Disease site. Now we will use an NCBI sequence data! A new page will appear identity for the top three matches? application called BLAST, Basic Local with the results of your search. What percentage of each aligned Alignment Search Tool. BLAST is an 4. On the search results page, below the sequence showed gaps indicating invaluable program for searching through Graphic Summary you will see a cat- sequence differences? GenBank and other databases to find egory called Descriptions and a table DNA- and protein-sequence similarities c.
    [Show full text]
  • The Pennsylvania State University Schreyer Honors College
    THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF FOOD SCIENCE CHARACTERIZATION OF THE SELECTION OF MULTIPLE START CODONS BASED ON VARIATIONS IN RIBOSOME BINDING SITE TRANSLATION INITIATION STRENGTH HANNAH JEPSEN-BURGER SPRING 2014 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Food Science with honors in Food Science Reviewed and approved* by the following: Thomas L. Richard Professor of Agricultural and Biological Engineering Thesis Supervisor Edward G. Dudley Associate Professor of Food Science Thesis Adviser *Signatures are on file in the Schreyer Honors College ABSTRACT Translation initiation is the process by which a ribosome selects an initia- tion point from within an mRNA strand and initiates the process of convert- ing genetic code into protein for the cell. Although alternative methods of eukaryotic translation initiation have been extensively cataloged, analogous alternative prokaryotic translation initiation mechanisms still remain largely unstudied. This research addresses the effects of multiple start codons within seven nucleotides of each other on protein expression using common cloning techniques. Plasmid DNA sequences were manipulated to observe these ef- fects. By varying the ribosome binding site translation initiation rate of each start codon and measuring the fluorescent output of two reporters, the research determined that these two start codons may individually be se- lected by the ribosome for translation, resulting in multiple discrete protein species. i Contents 1 Introduction 1 1.1 Problem . .1 1.2 Hypothesis . .3 1.3 Research Objective . .4 2 Literature Review 5 2.1 Translation Initiation in Prokaryotes and Eukaryotes . .5 2.1.1 Translation Initiation in Eukaryotes .
    [Show full text]
  • Genetic and Biochemical Analysis of the Interaction Between the Yeast
    GENETIC AND BIOCHEMICAL ANALYSIS OF THE INTERACTION BETWEEN THE YEAST FATTY ACID SYNTHESIS ENZYME YBR159W AND THE TRANSLATION INITIATION COMPLEX eIF2B By Christopher Michael Browne Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY In Biochemistry August, 2013 Nashville, Tennessee Approved: Professor Andrew J. Link Professor Kevin L. Schey Professor Claus Schneider Professor P. Anthony Weil Professor Nicholas J. Reiter This dissertation focuses on the biochemical and genetic characterization of the protein-protein interaction in the budding yeast Saccharomyces cerevisiae between the cytosolic translation initiation guanine nucleotide exchange factor eIF2B and the endoplasmic reticulum (ER) membrane-embedded very- long-chain fatty acid (VLCFA) synthesis beta-keto-reductase enzyme YBR159W (IFA38). The dissertation is divided between the physical characterization of the interaction and examination of the functional consequences the ybr159wΔ deletion has on the yeast cell’s physiology. I first look at how the interaction is occurring in yeast. I utilize yeast 2-hybrid analysis to show that eIF2B subunits GCD6 and GCD7 interact with YBR159W. My experiments show that eIF2B does not interact with other VLCFA synthesis enzymes and that YBR159W does not interact directly with the other canonical components of the eIF2B complex. Compared to a wild type strain, a ybr159wΔ null yeast strain has a reduced growth rate and the hallmarks of a reduced translation activity including reduced 35S-methionine incorporation and low levels of polyribosomes. It is unknown if the reduced translation rate is a direct or indirect consequence of the ybr159wΔ mutation.
    [Show full text]
  • Control of Translation Initiation and Neuronal Subcellular Localisation of Mrnas by G-Quadruplex Structures
    University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Centre for Biological Sciences Control of translation initiation and neuronal subcellular localisation of mRNAs by G-quadruplex structures by James Philip Robertson Schofield Thesis for the degree of Doctor of Philosophy 2015 i UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES CENTRE FOR BIOLOGICAL SCIENCES Doctor of Philosophy Control of translation initiation and neuronal subcellular localisation of mRNAs by G- quadruplex structures by James Philip Robertson Schofield The translation of mRNA is a key regulatory step in control of gene expression. The primary sequence of an mRNA determines much of the regulation of translation, including the location of translation initiation. The efficiency of translation initiation is determined by the initiation codon and its context. Translation initiation occurs at the canonical initiation codon, AUG, or a less efficient near canonical non-AUG alternative initiation codon (AIC).
    [Show full text]
  • Position-Dependent Codon Usage Bias in the Human Transcriptome
    bioRxiv preprint doi: https://doi.org/10.1101/2021.08.11.456006; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Position-dependent Codon Usage Bias in the Human Transcriptome Kaavya Subramanian1, Nathan Waugh2, Cole Shanks1, David A Hendrix1,2 1. School of Electrical Engineering and Computer Science, Oregon State University 2. Department of Biochemistry and Biophysics, Oregon State University bioRxiv preprint doi: https://doi.org/10.1101/2021.08.11.456006; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT All life depends on the reliable translation of RNA to protein according to complex interactions between translation machinery and RNA sequence features. While ribosomal occupancy and codon frequencies vary across coding regions, well-established metrics for computing coding potential of RNA do not capture such positional dependence. Here, we investigate position- dependent codon usage bias (PDCUB), which dynamically accounts for the position of protein- coding signals embedded within coding regions. We demonstrate the existence of PDCUB in the human transcriptome, and show that it can be used to predict translation-initiating codons with greater accuracy than other models. We further show that observed PDCUB is not accounted for by other common metrics, including position-dependent GC content, consensus sequences, and the presence of signal peptides in the translation product.
    [Show full text]
  • דוקטור לפילוסופיה Doctor of Philosophy
    עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By אורה חיימוב Ora Haimov חקר מנגנון התרגום הייחודי המתווך על-ידי TISU Elucidating the unique translation initiation mechanism directed by TISU element מנחה: :Advisor פרופ' רבקה דיקשטיין Prof.Rivka Dikstein כסלו תשע"ז December, 2016 Acknowledgments First and foremost, I would like to express my deep gratitude and appreciation to my advisor, Prof. Rivka Dikstein, for her excellent guidance, passion for science, critical and creative thinking, dedication, encouragement, support and mostly for being humane. Rivka, the things I learned from you will remain with me throughout my entire career. I would also like to thank my former and current colleagues for their scientific assistance, good advice and mostly for their friendship. Special thanks to Dr. Anat Bahat for being our ultimate staff scientist and a good friend, to Shaked Ashkenazi for her good spirit and contribution to the lab atmosphere, to Ana Tamarkin Ben-harush for many wonderful moments that I will always cherish, to Dr. Hadar Sinvani for collaboration and to Dr. Rafi Emmanuel for the initiation of the “TISU Project”. I owe many thanks to Anna Uzonyi and Adi Jacob, two young and talented scientists who contributed to this study. Last but not least, I wish to thank my family, especially to my parents, Mordechai and Geula, from whom I learned the value of knowledge and education, to my husband, Reuven, for his love, and to my four precious children, Ariel, Eitan, Naama and Yehonatan, who made this whole period challenging, meaningful and much more satisfying.
    [Show full text]