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Investigations Into the Function of Structural Isoforms of the Platelet Derived Growth Factor A-Chain

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Investigations Into the Function of Structural Isoforms of the Platelet Derived Growth Factor A-Chain Investigations into the function of structural isoforms of the Platelet Derived Growth Factor A-chain. Richard Alan Pollock Department of Biology Medawar Building University College London Gower Street London WC1E 6BT. A Thesis submitted to the University of London for the degree of Ph.D ProQuest Number: 10609352 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 10609352 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 Sister, Louise. Abstract. Platelet derived growth factor (PDGF) is a covalent dimer of A and B chains with the structure AB, AA or BB depending on its source. The A and B chains are related but distinct polypeptides encoded by separate genes. PDGF-A can be differentially spliced, allowing production of two mature proteins one of which (PDGF-Al) is longer than the other (PDGF-As) by 15 amino acids at its carboxyl terminus. PDGF-A homodimers are thoughtto be important in early development and, later, for regulating glial cell development in the central nervous system (CNS). In order to gain a clearer understanding of the role of PDGF in vivo it is important to define the functional significance of the different isoforms and their cellular sources and targets. This thesis describes my attempts to elucidate the functional differences between PDGF-As and PDGF-Al and to determine whether they are differentially expressed in CNS derived tissues. To investigate the functional differences between PDGF-As and PDGF-A l I introduced plasmid vectors directing the expression of the two isoforms into cultured Cos cells and subjected the expressed molecules to biochemical and immunocytochemical analysis. These studies provide evidence that PDGF-As is secreted as a freely diffusible molecule while PDGF-A l is sequestered near the cells that produce it by binding to heparin-like molecules on the cell surface and in the extracellular matrix (ECM). The implications of these findings are discussed with reference to other factors that exist in different isoforms that are either freely diffusible or immobilised at the cell surface and within the ECM. I attempted to analyse the expression pattern in the CNS of the two isoforms by a combination of techniques, including SI nuclease protection, PCR amplification on reverse transcribed RNA, cDNA cloning and sequencing. These studies suggest that PDGF-As predominates in the tissues and cell types I have examined in agreement with other studies of a similar nature. Acknowledgements I would like to thank my supervisor Dr. W. D. Richardson for help and guidance throughout my time in his lab and during the preparation of this thesis. I would also like to thank others in the lab and members of the biology department including Mike Mosely for making some of the expression vectors in the appendix, Dr. Nigel Pringle and Dr. Leon Nawrocki for their useful advice and for proof reading Chapter 2, Dr. Tessa Crompton for the invaluable and over-extended loan of her Mac, Dr. Bob Harris for his advice and virus club companionship, Dr. Barbara Barres for keeping me enthusiastic late into the night, Dr. Colin Hodgkinson for his music and dancing (sorry about the tupperware) and Georgina Stevens for her encouragement in the later stages. I would also like to thank my flatmate Adam Leibowitz for his consistent and oft amusing floccinaucinihilipilification during our years together in CamberHell and those who encouraged me when I needed it and put up with me the rest of the time including David, Robert, Karen, Zde, Anthony, Billy, Paul, Rab, and Jazz. Finally I would like to thank my grand parents, my sister and my mother and father whose support (financial and moral) and tireless encouragement throughout this, as with everything, has been beyond value. Thank you. List of Contents. Title p a g e ............................................................................................................................ 1 Dedication ............................................................................................................................2 Abstract ................................................................................................................................ 3 Acknowledgements ...........................................................................................................4 List of Contents ....................................................................................................................5 List of Figures ..................................................................................................................... 10 List of T ables........................................................................................................................16 Abbreviations ..................................................................................................................... 17 Chapter 1. Introduction ....................................................................................................20 1.1 Growth factors act by binding to cell surface receptors ...........................20 1.2 Growth factors can be grouped into families based on sequence similarity .................................................................................................21 A) Insulin and the insulin like growth factors (IGFs) ......................... 21 B) The epidermal growth factor (EGF) family ...................................... 22 C) The transforming growth factor beta (TGF-p) family ....................23 D) The fibroblast growth factor (FGF) family ...................................... 25 The fibroblast growth factor receptor (FGFr) family............................................................................................... 27 Fibroblast growth factors and heparin binding ......................29 E) The hemopoietic and neuropoietic growth factors ........................32 F) The platelet derived growth factor (PDGF) family ......................... 37 The role of PDGF in the vascular system ..................................37 Abnormal PDGF activity and proliferative disease .................38 PDGF and Neoplasia ...................................................................... 39 PDGF in development ................................................................... 40 Alternative splicing of PDGF A-chain mRNA ............................42 1.3 Aims of thesis ....................................................................................................44 Chapter 2. Materials and Methods ................................................................................49 2.1 Bacteriology ......................................................................................................49 A) Bacterial strains .....................................................................................49 B) Growth media and agar plates ............................................................49 C) Preparation of competent bacteria ....................................................50 i) Calcium chloride method .......................................................50 ii) Hexamine cobalt chloride method ........................................ 50 D) Transformation of bacteria with DNA ...............................................50 E) Long-term storage of recombinant bacteria .....................................51 2.2 Molecular biology ............................................................................................ 51 A) Phenol/chloroform extractions ...........................................................51 B) Ethanol precipitation of nucleic acids ...............................................51 C) Restriction digest of DNA ..................................................................... 52 D) End labelling of DNA fragments .........................................................52 E) Dephosphorylation of DNA termini ................................................... 52 F) Ligation of DNA ....................................................................................... 53 G) Screening plasmid DNA from transformed bacterial colonies .......................................................................................................... 53 i) Plasmid mini preparation ......................................................... 53 ii) Transformed bacterial colony lifts and hybridisation ....................................................................................53 H) "Maxi" plasmid preparation ................................................................ 54 I) Preparation of genomic DNA ................................................................ 55 J) Preparation of RNA ................................................................................. 55 K) Poly (A)+ selection ................................................................................. 56 L) Electrophoretic analysis of nucleic acids .......................................... 56 i) Agarose gels ..................................................................................56
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