Chromosome Structural Analysis

Transcription

1 Chromosome Structural Analysis A Practical Approach Edited by WENDY A. BICKMORE Cell Genetics Section, MRC Human Genetics Unit, Western General Hospital, Edinburgh OXJORD UNIVERSITY PRESS 1999

2 List of contributors Abbreviations 1. Mapping protein/dna interactions in vivo using ligation-mediated polymerase chain reaction 1 Donald Macleod 1. Introduction 1 Methods used in determining protein/dna interactions in vitro 1 Determining protein/dna interactions in vivo 2 2. The ligation-mediated polymerase chain reaction (LMPCR) 3 Applications of LMPCR 4 3. Mapping protein factor binding sites in vivo with LMPCR and DMS 5 DNA modification by DMS in vitro 5 DMS modification of DNA in vivo 7 Amplification of DMS/piperidine-cleaved DNA by LMPCR 7 Analysis of LMPCR reactions 9 4. Mapping nucleosomes using micrococcal nuclease and LMPCR 13 Isolation of nuclei from cultured cells 13 Preparation of DNA from nuclei treated with MNase 14 Cleavage of genomic DNA with MNase 16 Acknowledgements 18 References Mapping DNA target sites of chromatinassociated proteins by formaldehyde cross-linking in Drosophila embryos 21 Giacomo Cavalli, Valerio Orlando, and Renato Paro 1. Introduction Outline of the method 22

3 3. Formaldehyde cross-linking in staged Drosophila embryos 23 Preparation offlycages and collection of staged embryos 23 Optimizing cross-linking conditions Immunoprecipitation of cross-linked embryonic chromatin and PCR amplification of the immunoprecipitated DNA Analysing the enrichment of putative target sequences in the PCR-amplified DNA 31 Slot-blot analysis of the enrichment of putative PC target sequences 31 Mapping DNA target sites for Polycomb and GAGA factor in the Drosophila bithorax complex Concluding remarks 36 References Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP) 39 Karl Ekwall and Janet F. Partridge 1. Introduction Fluorescence in situ hybridization (FISH) analysis of fission yeast 40 Preparation of probes 40 Cell fixation and cell-wall digestion Chromatin immunoprecipitation from fission yeast 48 Fixation of yeast cells to maintain protein localization 49 Preparation of chromatin extract 50 Immunoprecipitation of chromatin ' 51 Analysis of immunoprecipitated DNA sequences 55 Acknowledgements 56 References Isolation of vertebrate metaphase chromosomes and their analysis by FISH 59 Jeff Craig 1. Introduction General equipment required for FISH 59 xii

4 3. Production of metaphase chromosomes as substrates for FISH Production of fixed metaphase chromosome spreads Production of long prometaphase chromosomes Isolation of suspensions of unfixed metaphase chromosomes 4. Spreading fixed chromosomes 5. Pretreatments of slides Pretreatment of mitotic chromosome spreads Salt extraction of isolated metaphase chromosomes 6. Labelling DNA probes Choice of label Nick translation Random priming Labelling by PCR Quantifying label incorporation 7. Hybridization Preparation of probes and slides Hybridization 8. Detecting hybridized probe 9. Counterstaining and mounting Simple counterstaining Chromosome banding Acknowledgements References Studying progression of vertebrate chromosomes through mitosis by immunofluorescence and FISH Beth A. Sullivan and Peter E. Warburton 1. Introduction Fundamental aspects of mitosis 81 The mitotic spindle 81 Chromosomes Detecting centromere/kinetochore proteins on metaphase chromosomes In situ hybridization following immunofluorescence The use of anti-mitotic drugs 90 Colcemid/colchicine 90 Nocodazole 91 xiii si

5 Vinblastine and other drugs 91 Cytochalasin/dihydrocytochalasin B (DCB) Immunofluorescence on anaphase and telophase cells/chromosomes 92 C-anaphase: sister chromatid separation and anaphase in the presence of Colcemid 92 Anaphase chromosomes visualized on the mitotic spindle 94 Anaphase studies on cytokinesis-blocked cells 96 References 100 Analysis of mammalian interphase chromosomes by FISH and immunofluorescence 103 Joanna M. Bridger and Peter Lichter 1. Introduction Preparation of sample material 105 Adherent cells 105 Suspension cells 106 DNA halo preparations 106 Fixation and permeabilization 107 Improving probe penetration Probes 111 Chromosomal painting probes 111 Probe labelling Fluorescence in situ hybridization 113 Denaturation 113 Hybridization Washing 115 Detection of reporter molecules Immunofluorescence in combination with FISH 116 Primary and secondary antibody incubations after FISH 118 Primary antibody incubation predenaturation and secondary antibody incubation postdenaturation 119 Primary and secondary antibody incubations prior to FISH denaturation 119 Reporter-conjugated primary antibody incubated predenaturation Mounting the slides Analysis 120 References 121 xiv

6 7. Fluorescence in situ hybridization in whole-mount tissues 125 Abby F. Dernburg 1. Introduction Probe synthesis and labelling 127 General considerations 127 Why use fluorescence-based detection? 127 Choice of labelling and detection reagents 128 Probe synthesis Fixation methods for whole-mount FISH Hybridization methods Troubleshooting Microscopy and image analysis Future directions 144 References 144 Acknowledgement Analysing the substructure of mammalian nuclei, in vitro Dean A. Jackson 1. Introduction The nuclear matrix and nucleoskeleton Methods used to analyse nuclear organization 148 The nuclear matrix 148 Nucleoids. 149 The nuclear scaffold 150 The 'low-salt' nuclear matrix 151 The nucleoskeleton Studying the chromatin loops of different nuclear derivatives 153 Chromatin loops after hypertonic or hypotonic treatment 153 Chromatin loops under 'physiological' conditions 154 Technical tips on cutting and electroeluting chromatin 156 The frequency and nature of attachment sites in different nuclear derivatives The morphology of different nuclear derivatives Assaying nuclear function and nuclear proteins in permeabilized cells 160 Labelling sites of replication and transcription in vitro 160 Technical tips on labelling sites of replication and transcription 161 xv ui

7 Studying protein distribution relative to sites of transcription or replication in permeabilized cells. 162 A typical example Conclusions 164 References Chromosome assembly in vitro using Xenopus egg extracts Jason R. Swedlow 1. Introduction Chromosome structure and biochemistry Preparation of Xenopus egg extracts for chromatin and chromosome assembly in vitro 168 Xenopus egg maturation 168 Xenopus egg extracts 168 Chromosome assembly extracts technical tips Chromatin and chromosome assembly in vitro 175 Assembly and isolation of chromatin and chromosomes technical tips Immunofluorescence of in vitro assembled chromosomes Functional analysis of the role of specific proteins in chromatin and chromosome structure by immunodepletion 180 Acknowledgements 181 References Chromosome fragmentation in vertebrate cell lines 183 Christine J. Farr 1. Introduction Telomere-associated chromosome fragmentation Experimentally induced de novo telomere formation 185 Design of the telomere-seeding construct 185 Transfection of the telomere-seeding construct 186 Screening stable transfectants for de novo telomere formation Targeted de novo telomere formation Targeted truncation events in the recombination-proficient avian cell line DT xvi ui

8 6. The characterization of chromosomes modified by de novo telomere formation and fragmentation 193 Estimation of minichromosome size 194 Assays for mitotic stability of minichromosomes Concluding remarks 197 References 197 Al. List of suppliers 199 Index 207 XVll