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IDENTIFICATION of the Sm N PROTEIN and STUDIES on ITS EXPRESSION Neil Grey Sharpe Department of Biology University College Londo

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IDENTIFICATION of the Sm N PROTEIN and STUDIES on ITS EXPRESSION Neil Grey Sharpe Department of Biology University College Londo IDENTIFICATION OF THE Sm N PROTEIN AND STUDIES ON ITS EXPRESSION Neil Grey Sharpe Department of Biology University College London A thesis submitted for the degree of Doctor of Philosophy University of London, 1990 1 ProQuest Number: 10629533 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10629533 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 To my parents 2 Acknowledgements I would primarily like to thank David Latchman for his skilled and considerate supervision throughout the course of this research. I am grateful to the members of both the Zoology Department, UCL and the Medical Molecular Biology Unit for their advice and support; in particular, John Estridge, Kyung-Lib Jang, Baljinder Mankoo, Mukesh Patel, Annie Rowe, Bob Harris, Janet Partridge, Karen Lillycrop and Aviva Symes. I am especially grateful to Lynn Kemp for her invaluable tuition in the initial stages of this research. In addition, I extend my gratitude to David Williams and Juliet Roberts for their comments on the text of this thesis. Abstract The major species of mammalian Sm proteins associate with snRNA molecules to form small nuclear ribonucleoprotein particles (snRNPs) which are essential for pre-mRNA splicing. A series of immunoblots were probed with an anti-Sm monoclonal antibody. These experiments led to the identification of a cell-specific 28kDa protein called Sm N. The expression of the Sm N protein is restricted to cell lines and tissues which have the ability to utilise the alternative splicing pathway of the calcitonin/CGRP gene. This correlation suggests that the Sm N protein may play a role in determining the use of this alternative splicing pathway. A Sm N cDNA clone was isolated by immunoscreening a HeLa Xgtll expression library. Characterisation of the cDNA clone showed that the Sm N protein consists of 240 amino acids. It has a proline-rich carboxyl terminus and it is closely related to the Sm B and B' proteins. Northern blot analysis revealed that the Sm N protein is encoded by a 1.6kb mRNA transcript. RNA analysis showed that the Sm N gene is differentially expressed in HeLa cell lines. The levels of the Sm N protein and mRNA were shown to decline during the differentiation of embryonal carcinoma stem cells to parietal endoderm-like cells. Furthermore, the levels of the Sm N protein decline during the differentiation of embryonal stem cells suggesting that this effect may occur in vivo. The sera of some patients with the autoimmune disease systemic lupus erythematosus (SLE) contain anti-Sm autoantibodies. The anti-Sm monoclonal antibody which was used to identify Sm N recognises a disease autoepitope. In order to localise this epitope on the Sm N protein, a short, 135 nucleotide Sm N clone was obtained by immunoscreening a kgtll expression library. A lysogen of this clone was used to detect the presence of anti-Sm antibodies in SLE sera. It was found that both anti-Sm and anti-RNP autoantibodies reacted with the fusion protein. This effect was due to amino add sequence similarities between Sm N and the U1 snRNP-spedfic C protein. Immunoblotting analysis was used to show that the highly immunoreactive Sm B, B', and D proteins increase in abundance in Vero cells infected with herpes simplex virus type 2. This effect may increase the antigenicity of these Sm proteins. 4 Abbreviations The abbreviations which are used in this thesis are those described in the 'Policy of the Journal and Instructions to Authors' of the Biochemistry Journal (Biochem. J. (1989) 257 1-21). In addition, the following abbreviations have been used: bp base pairs BSA • bovine serum albumin cAMP cyclic adenosine monophosphate CGRP calcitonin gene related peptide DTT dithiolthreitol EC embryonal carcinoma EDTA ethylenediaminetetra-acetic acid ES embryonal stem HSV herpes simplex virus IPTG isopropyl P-D-thiogalactopyranoside kb kilobase kDa. kilodalton MTCD Mixed connective tissue disease NEPHGE Nonequilibrium pH gradient electrophoresis PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline P i post infection RA retinoic acid RNP ribonucleoprotein particle SDS sodium dodecyl sulphate SLE Systemic lupus erythematosus snRN A small nuclear RNA snRNP small nuclear ribonucleoprotein particle TEMED N,N,N',N' - tetramethylethylenediamine UCL University College London UCMSM University College and Middlesex School of Medicine X-gal 5-bromo-4-chloro-3-indolyl-p-galactopyranoside NIMR National Institute for Medical Research ICRF Imperial Cancer Research Fund 5 Table of Contents TITLE PA G E.................................................................................................... 1 DEDICATION.................................................................................................. 2 ACKNOWLEDGEMENTS............................................................................. 3 ABSTRACT....................................................................................................... 4 ABBREVIATIONS........................................................................................... 5 TABLE OF CONTENTS.................................................................................. 6 LIST OF FIGURES............................................................................................ 10 LIST OF TABLES.............................................................................................. 12 CHAPTER 1: INTRODUCTION....................................................................13 The Structure of snRNPs .....................................................................14 Pre-mRNA Splicing..............................................................................19 Role of snRNPs in Pre-mRNA Splicing......................................... 20 The Involvement of snRNPs in the Spliceosome........................ 21 Alternative Pre-mRNA Splicing.......................................................25 Czs-Regulation of Alternative Splice Site Selection .................... 26 Traws-Regulation of Alternative Splicing .......................................28 The Possible Nature of Cell Specific Trans-acting Alternative Splicing Factors.............................................................. 33 Anti-snRNP Autoantibodies and Systemic Lupus Erythematosus ...................................................................................... 34 The Aetiology of SLE ...........................................................................35 The Aetiology of Autoantibodies in SLE ........................................ 36 CHAPTER 2: MATERIALS AND METHODS............................................39 Materials................................................................................................ 40 Bacterial Strains........................................................................ 40 Cell Lines......................................................................................40 Sera and Monoclonal Antibodies .......................................... 41 Viruses .......................................................................................... 41 DNA............................................................................................ 41 Protein Samples...........................................................................42 Enzymes ........................................................................................42 Radiochemicals.......................................................................... 42 Other Materials & Reagents......................................................42 Buffers, Solutions and Growth Media ....................................43 6 M ethods.................................................................................................. 44 DNA.................................................................................................... 44 Immunoscreening X gt 11 libraries ........................................ 44 Screening Xgt 11 libraries with radiolabelled DNA probes.............................................................................................45 Determination of phage titre.....................................................45 Isolation of bacteriophage DNA............................................. 46 Creating recombinant lysogens .............................................. 46 Restriction enzyme analysis ...................................................... 46 Gel Electrophoresis of DNA......................................................46 Isolation of DNA fragments.....................................................47 Construction of recombinant plasmids ................................ 47 Isolation of plasmid DNA....................................................... 49 Southern blotting ....................................................................... 50 Radiolabelling of double-stranded DNA fragments 51 DNA sequencing ....................................................................... 51 DNA and Protein sequence analysis ....................................
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