Sequence specific visualization of DNA in live mammalian cells [Elektronische Ressource] / by Giulia Mearini
108 Pages
English
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Sequence specific visualization of DNA in live mammalian cells [Elektronische Ressource] / by Giulia Mearini

Downloading requires you to have access to the YouScribe library
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108 Pages
English

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Published 01 January 2005
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Language English
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Sequence specific visualization of DNA
in live mammalian cells
A Dissertation
Submitted for the Doctoral Degree
Department of Biology
University of Hamburg
by
Giulia Mearini
from Italy
Hamburg, 2005The present dissertation was prepared in the time from March 2001 until
October 2004 at the department of Molecular Cell Biology of the Heinrich
Pette Institute for Experimental Virology and Immunology at the University of
Hamburg.Acknowledgements
I would like to express my deep gratitude to Dr. Frank O. Fackelmayer for giving
me the opportunity to make the PhD in his group, for helpful suggestions and
constant discussions during the development of my project, and for the relaxed
working atmosphere. I thank Prof. Dr. P.E. Nielsen (Copenhagen) for design and
synthesis of PNA molecules.
A very special thanks goes to my colleagues Maike Bossert, Andrea Schwan
der, Frank (jr.) Herrmann and Roger Helbig for help in the lab (and not only!), the
extreme patience with my German and the many nice hours we spent together.
To Maike and Andrea my gratitude for constant support and help: without them
I wouldn’t be so well integrated in Germany now.
Thanks also to the many nice people of Deppert’s department in particular
Rudolph Reimer for ’tips and tricks’ in microscopy, Heike Helmbold and Daniel
Speidel for moral support and friendship.
I thank my parents, my sister Claudia, my best friend Silvia Masciarelli
and Elena Mattia for the encouragement in coming here in Germany and the
permanent support in good and bad times.
In Hamburg I found many nice poeple, among them Ulrike Sonntag Kroll
who I thank for her hospitality, help and friendship, and ’il mio tesoro’ Christian
Hermsdorf. I thank him for staying always at my side, for making me laugh and
Afor introducing me to LT X world.E
Last, but not least, I would like to thank Prof. Dr. Wolfgang Deppert for the
use of the microinjection system and the supervision of the present work.
In the first two years I was supported by a personal fellowship of the Istituto
Pasteur, Fondazione Cenci Bolognetti, University of Rome ’La Sapienza’.Contents
List of Figures V
List of Tables VII
Abbreviations VIII
1 Introduction 1
1.1 The cell nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Nuclear architecture . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1.1 Nuclear compartments . . . . . . . . . . . . . . . . 2
1.1.1.2 DNA organization . . . . . . . . . . . . . . . . . . . 3
1.1.1.3 Chromosome territories . . . . . . . . . . . . . . . 4
1.1.1.4 The nuclear matrix . . . . . . . . . . . . . . . . . . 5
1.2 Scaffold/Matrix Attachment Regions (S/MAR) . . . . . . . . . . . . 6
1.2.1 S/MAR binding proteins . . . . . . . . . . . . . . . . . . . . 7
1.3 The nucleus is a dynamic organelle . . . . . . . . . . . . . . . . . . 8
1.3.1 Fluorescence imaging methods . . . . . . . . . . . . . . . . 8
1.3.2 Protein dynamics . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.3 Nucleic acids dynamics . . . . . . . . . . . . . . . . . . . . . 10
1.3.4 Subcompartment dynamics . . . . . . . . . . . . . . . . . . 10
1.3.5 Dynamics and transcription . . . . . . . . . . . . . . . . . . 11
1.4 In vivo visualization of DNA . . . . . . . . . . . . . . . . . . . . . . . 11
1.5 Peptide nucleic acids (PNA) . . . . . . . . . . . . . . . . . . . . . . . 13
1.5.1 Structure and properties . . . . . . . . . . . . . . . . . . . . 13
1.5.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.6 Lac operator/repressor system . . . . . . . . . . . . . . . . . . . . . 15
2 Aims of the work 17Contents III
3 Materials and methods 18
3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1.1 Devices, chemicals and equipment . . . . . . . . . . . . . . 18
3.1.2 Buffers and solutions . . . . . . . . . . . . . . . . . . . . . . 21
3.1.3 Oligos and primers . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.4 Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.1 General methods . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.2 Cloning a binding site for PNA . . . . . . . . . . . . . . . . . 23
3.2.3 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.4 Labeling of plasmid DNA by PNA . . . . . . . . . . . . . . . 24
3.2.4.1 Purification and concentration of PNA/DNA com
plexes . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.5 Electrophoretic mobility shift assay . . . . . . . . . . . . . . 24
3.2.6 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.7 Transfection of eukaryotic cells . . . . . . . . . . . . . . . . 25
3.2.7.1 Transfection with polyethylenimine (PEI) . . . . . 25
3.2.7.2 T by electroporation . . . . . . . . . . . 26
3.2.8 Microinjection of COS 7 . . . . . . . . . . . . . . . . . . . . . 26
3.2.9 Sequential extraction of cells . . . . . . . . . . . . . . . . . . 27
3.2.10 Hirt extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.11 Real time PCR (RT PCR) . . . . . . . . . . . . . . . . . . . . 28
3.2.12 Establishment of stable cell lines . . . . . . . . . . . . . . . 28
3.2.13 Fixation and staining of cells for FACS analysis . . . . . . . 29
3.2.14 Preparation of fixed specimens for confocal microscopy . . 30
3.2.14.1 Analysis of fixed cells . . . . . . . . . . . . . . . . . 31
3.2.14.2 Fluorescence Recovery After Photobleaching (FRAP) 31
3.2.14.3 4D life cell imaging . . . . . . . . . . . . . . . . . . 34
4 Results 38
4.1 Labeling of plasmid DNA by peptide nucleic acid (PNA) . . . . . . . 38
4.1.1 Purification and concentration of PNA/DNA complexes . . 39
4.2 How to get PNA/DNA complexes into nuclei? . . . . . . . . . . . . . 40Contents IV
4.3 Microinjection is not toxic for the cells . . . . . . . . . . . . . . . . . 40
4.4 Nuclear localization is due to plasmid component of PNA/DNA
complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.5 The final localization of an injected plasmid is reached after hours 43
4.6 Nuclear of plasmid DNA . . . . . . . . . . . . . . . . . . 45
4.7 Subnuclear localization of PNA/DNA complexes . . . . . . . . . . . 48
4.8 Plasmid DNA is resistant toward detergent and high salt extraction 50
4.8.1 Quantification of plasmid DNA bound to nuclear scaffold . 51
4.9 Dynamics of plasmid DNA in vivo . . . . . . . . . . . . . . . . . . . 52
4.10 Establishment of CHO lacOp/lacI GFP stable cell line . . . . . . . 57
4.11 The integration site of lac operator . . . . . . . . . . . . . . . . . . . 58
4.12 FACS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.13 Lac repressor remains bound to its target sequence throughout
mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.14 Mobility of chromatin loci in vivo . . . . . . . . . . . . . . . . . . . . 62
4.14.1 Chromatin mobility in tetraploid cells . . . . . . . . . . . . 67
4.15 FRAP analysis for lacI GFP . . . . . . . . . . . . . . . . . . . . . . . 68
5 Discussion 71
5.1 Localization of small circular DNA . . . . . . . . . . . . . . . . . . . 72
5.2 Dynamics of plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3 of chromatin loci in vivo . . . . . . . . . . . . . . . . . . . 78
6 Summary 83
Bibliography 84List of Figures
1.1 Nuclear architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Chemical structure of PNA and triplex formation . . . . . . . . . . . . 13
3.1 Fluorescence Recovery After Photobleaching (FRAP) . . . . . . . . . . 32
3.2 Plot of normalized FRAP data . . . . . . . . . . . . . . . . . . . . . . . . 34
4.1 Schematic representation of PNA/DNA complex . . . . . . . . . . . . . 38
4.2 Determination of optimal PNA:DNA molar ratio for complex formation 39
4.3 Purification of PNA/DNA complexes . . . . . . . . . . . . . . . . . . . . 40
4.4 Comparison of amount of microinjected plasmid . . . . . . . . . . . . 41
4.5 Cell viability after microinjection . . . . . . . . . . . . . . . . . . . . . . 41
4.6 GFP expression from PNA labeled plasmid . . . . . . . . . . . . . . . . 42
4.7 Microinjection of free PNA with FITC dextran . . . . . . . . . . . . . . 43
4.8 Time course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.9 Stability of PNA/DNA complexes localization . . . . . . . . . . . . . . . 44
4.10 Nuclear localization of plasmid DNA in live cells . . . . . . . . . . . . . 45
4.11 of DNA in fixed cells . . . . . . . . . . . . 46
4.12 Localization of control plasmids . . . . . . . . . . . . . . . . . . . . . . 47
4.13 of plasmid DNA is independent of its sequence . . . . . . 47
4.14 Co microinjection of PNA/DNA complexes with expression vector for
hFibrillarin protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.15 Co microinjection of PNA/DNA complexes with expression vector for
PML protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.16 Co microinjection of PNA/DNA complexes with expression vector for
SAF A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.17 Sequential extraction of COS 7 cells . . . . . . . . . . . . . . . . . . . . 50
4.18 DNA loops and anchored plasmid DNA after high salt extraction . . . 51
4.19 Resistance of transfected toward high salt . . . . . 52List of Figures VI
4.20 FRAP experiment with pMII plasmid . . . . . . . . . . . . . . . . . . . . 53
4.21 FRAP with pEPI 1 plasmid . . . . . . . . . . . . . . . . . . 53
4.22 FRAP experiment with pK2 plasmid . . . . . . . . . . . . . . . . . . . . 54
4.23 FRAP with pMII linear plasmid . . . . . . . . . . . . . . . . 54
4.24 Re elaborated curves and mobile fractions . . . . . . . . . . . . . . . . 55
4.25 FRAP experiments forfl Gal NLS and LaminB1 . . . . . . . . . . . . . 56
4.26 Stable lacOp/lacI GFP cell clones . . . . . . . . . . . . . . . . . . . . . 58
4.27 Doublets on chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.28 Flow cytometry histograms of CHO lacOp/lacI GFP subclones . . . . 60
4.29 Persistance of lacI GFP DNA bindig during mitosis . . . . . . . . . . . 61
4.30 DRB treatment is effective . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.31 Paths and frequency histograms for spots in the nuclear interior . . . 63
4.32 Paths and fr for spots at the nuclear periphery . 64
4.33 Density plots of distances between two time frames . . . . . . . . . . . 65
› fi
24.34 Mean square change in distance ¢d . . . . . . . . . . . . . . . . . . 66
4.35 Fluctuation in distances between two spots . . . . . . . . . . . . . . . 67
› fi
24.36 Mean square change in distance ¢d between two spots . . . . . . 68
4.37 FRAP of nucleoplasmic lacI GFP . . . . . . . . . . . . . . . . . . . . . . 69
4.38 FRAP of lacI GFP at the spot . . . . . . . . . . . . . . . . . . . . . . . . 69
5.1 Interpretation of fluorescence recovery curve and mobile fractions . . 77