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Functional Characterisation of the Spindle Pole Body Component Bbplp

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Functional Characterisation of the Spindle Pole Body Component Bbplp Functional Characterisation of the Spindle Pole Body Component Bbplp Carolin Schramm A thesis submitted for the degree of Doctor of Philosophy University of Glasgow Faculty of Medicine & Beatson Institute for Cancer Research Glasgow January 2001 ProQuest Number: 13818806 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 13818806 Published by ProQuest LLC(2018). 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 I'KQ'E, K U 9 t ‘D ‘DO % A GLASGOW u n iv e r sit y ILIBRARY: o o P i \ CONTENTS I Contents C o n t e n t s _____________________________________________________________ I List of Tables__________________________________________________________VI List of Figures_______________________________________________________ VII List of Publications____________________________________________________ IX Abbreviations __________________________________________________________ X Summary____________________________________________________________ XIII 1. Introduction_________________________________________________________1 1.1 Events in the S. cerevsiae cell cycle____________________________________ 1 1.2 Cell cycle regulation and cell cycle checkpoints inS. cerevisiae______________ 3 1.3 Functions of the cytoskeleton throughout the cell cycle____________________ 9 1.3.1 The microtubule cytoskeleton in the cell c y c_______________________________________ le 9 1.3.2 The actin cytoskeleton in the cell cycle_____________________________________________ 10 1.4 Microtubule organising centres______________________________________ 13 1.4.1 The centrosome_________________________________________________________________ 14 1.4.2 The spindle pole body (SPB)______________________________________________________ 17 1.4.2.1 Morphology of the SPB______________________________________________________ 17 1.4.2.2 Components of the SPB______________________________________________________ 18 1.4.2.2.1 Proteins forming inner and central regions of the SPB________________________18 1.4.2.2.2 Components of the outer plaque_____________________________ 21 1.4.2.2.3 Components which associate with SPBs and centromeres____________________ 23 1.4.2.2.4 Components which are shared by SPBs and nuclear pores____________________ 24 1.4.2.2.5 Proteins of the bridge structure____________________________________________25 1.4.2.2.6 Components of the satellite ______________________________________________ 26 1.4.2.2.7 Further proteins associated with SPB s _____________________________________ 27 1.4.3 Homologous proteins of MTOCs___________________________________________________28 1.4.4 The function of MTOCs__________________________________________________________ 29 1.4.5 Duplication of M TOCs__________________________________________________________ 31 1.4.5.1 Duplication of centrosomes__________________________________________________ 32 1.4.5.2 Duplication of the budding yeast SPB __________________________________________32 1.4.5.2.1 Duplication process_____________________________________________________32 1.4.5.2.2 Proteins involved in SPB duplication______________________________________ 34 1.4.5.2.3 Involvement of theP K C l MAP kinase pathway in SPB duplication ___________ 37 1.4.5.3 Similarities of MTOC duplication in different organisms_________________________37 1.5 Aim of thesis: characterisation of Bbplp______________________________ 39 2. Materials and Methods ______________________________________________ 41 2.1 Materials ________________________________________________________ 41 2.1.1 Chemicals _______________________________________________________________________41 2.1.2 Buffers_________________________________________________________________________ 41 2.1.3 Enzymes________________________________________________________________________42 2.1.4 Kits____________________________________________________________________________ 43 2.1.5 Standards _______________________________________________________________________43 2.1.5.1 DNA standards ______________________________________________________________43 2.1.5.2 Protein standards ___________________________________________________________ 43 2.1.6 Primer__________________________________________________________________________ 44 2.1.7 Plasmids ___________ 46 CONTENTS II 2.1.8 E. coli strains ___________________________________________________________________ 48 2.1.9 Yeast strains ____________________________________________________________________ 49 2.1.10 Antibodies____________________________________________________________________ 50 2.1.11 Media _________________________________________________________________________51 2.1.11.1 Media for yeast____________________________________________________________ 51 2.1.11.2 Media for bacteria _________________________________________________________ 54 2.1.12 Filters and membranes __________________________________________________________ 55 2.1.13 Photographic material __________________________________________________________ 55 2.1.14 Equipment____________________________________________________________________ 55 2.1.15 Software_______________________________________________________________________58 2.1.16 Internet services ________________________________________________________________ 58 2.2 Methods________________________________________________________ 59 2.2.1 Polymerase chain reaction (PCR) _________________________________________________ 59 2.2.1.1 Amplification of genes_______________________________________________________ 59 2.2.1.2 Colony PCR________________________________________________________________ 60 2.2.1.3 Mutagenic PCR _____________________________________________________________61 2.2.1.4 Amplification of cassettes____________________________________________________61 2.2.2 Precipitation of DNA_____________________________________________________________62 2.2.3 Restriction digest________________________________________________________________ 62 2.2.4 Gel electrophoresis_______________________________________________________________ 63 2.2.4.1 Separation of DNA__________________________________________________________ 63 2.2.4.2 Separation of proteins (SDS-PAGE)___________________________________________63 2.2.5 Staining techniques ______________________________________________________________64 2.2.5.1 Staining of DNA_____________________________________________________________64 2.2.5.2 Staining of proteins_________________________________________________________ 64 2.2.5.2.1 Coomassie Blue staining_________________________________________________ 64 2.2.5.2.2 Fairbanks staining _______________________________________________________ 65 2.2.5.2.3 Silver staining __________________________________________________________ 65 2.2.6 Extraction of DNA from agarose g e ls______________________________________________ 66 2.2.7 Culture of strains ________________________________________________________________ 66 2.2.7.1 Culture of E. coli strains ______________________________________________________ 66 2.2.1.2 Culture of yeast strains_______________________________________________________ 66 2.2.8 Cloning_________________________________________________________________________ 67 2.2.8.1 Preparation of vectors _______________________________________________________ 67 2.2.8.2 Preparation of inserts________________________________________________________ 68 2.2.8.3 Ligation___________________________________________________________________ 69 2.2.8.4 Competent c e lls_____________________________________________________________69 2.2.8.4.1 CompetentE.coli cells___________________________________________________69 2.2.8.4.2 Competent yeast cells____________________________________________________70 2.2.8.5 Transformation______________________________________________________________70 2.2.8.5.1 Transformation of bacteria cells ___________________________________________70 2.2.8.5.2 Transformation of yeast cells_____________________________________________ 71 2.2.9 Freezer stocks___________________________________________________________________ 71 2.2.9.1 Freezer stocks ofE. coli strains _______________________________________________ 71 2.2.92 Freezer stocks of yeast strains________________________________________________ 72 2.2.10 Isolation of D N A_______________________________________________________________72 2.2.10.1 Isolation of plasmid DNA fromE. c o li_______________________________________ 72 2.2.10.2 Isolation of plasmid DNA from yeast (plasmid rescue)__________________________ 72 2.2.10.3 Isolation of chromosomal DNA from yeast____________________________________ 73 2.2.11 Measurement of DNA concentration______________________________________________ 74 2.2.12 Measurement of protein concentrations____________________________________________ 74 2.2.13 Western blotting________________________________________________________________ 74 2.2.14 Automated sequencing__________________________________________________________ 75 2.2.15 FACS analysis _________________________________________________________________
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