Chromosome Structural Analysis A Practical Approach

by Bickmore, Wendy A.

• ISBN13: 9780199636983
• ISBN10: 0199636982
• Format: Paperback
• Copyright: 1999-07-29
• Publisher: Oxford University Press

Summary

The DNA of eukaryotes is packaged into chromosomes - each chromosomeconsisting of a very long molecule of DNA and various proteins (e.g. histones),and the number of chromosomes being characteristic for the species concerned.Chromosome analysis can provide a great deal of information for many aspects ofcellular genetics such as DNA replication, protein:DNA interactions and geneticmanipulation. The book is structured in a methodical fashion - the introductorychapters are centred around analysis of chromatin with chapters on the mappingof protein:DNA interactions in vivo using ligation-mediated PCR and the mappingof chromatin-associated proteins by formaldehyde cross-linking. The nextchapters concentrate on the study of whole chromosome structure, including:fission yeast chromosome analysis using FISH and CHIP, isolation of vertebratemetaphase chromosomes and their analysis by FISH, the study of vertebratechromosome progression through mitosis, and the analysis of mammalian interphasechromosomes by immunofluorescence and FISH. There then follow chapters on FISHin whole-mount tissues and the analysis of the sub-structure of mammalian nucleiin vitro. The final two chapters deal with the experimental manipulation ofchromosome structure, including: chromosome assembly in vitro using Xenopus eggextracts and chromosome fragmentation in vertebrate cell lines. Thiscomprehensive and informative laboratory manual includes a diverse range ofexperimental models for the analysis of chromosomes - such as vertebrates,Drosophila, yeast and Xenopus. Fully illustrated, it focuses on moderntechniques and approaches to the study of chromosome structure and will beinvaluable to researchers and academic staff in genetics, biomedical science andmolecular biology.

Table of Contents

List of Contributors	xix
Abbreviations	xxi
Mapping protein/DNA interactions in vivo using ligation-mediated polymerase chain reaction	1	(20)
Donald Macleod
Introduction	1	(2)
Methods used in determining protein/DNA interactions in vitro	1	(1)
Determining protein/DNA interactions in vivo	2	(1)
The ligation-mediated polymerase chain reaction (LMPCR)	3	(2)
Applications of LMPCR	4	(1)
Mapping protein factor binding sites in vivo with LMPCR and DMS	5	(8)
DNA modification by DMS in vitro	5	(2)
DMS modification of DNA in vivo	7	(1)
Amplification of DMS/piperidine-cleaved DNA by LMPCR	7	(2)
Analysis of LMPCR reactions	9	(4)
Mapping nucleosomes using micrococcal nuclease and LMPCR	13	(8)
Isolation of nuclei from cultured cells	13	(1)
Preparation of DNA from nuclei treated with MNase	14	(2)
Cleavage of genomic DNA with MNase	16	(2)
Acknowledgements	18	(1)
References	18	(3)
Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos	21	(18)
Giacomo Cavalli
Valerio Orlando
Renato Paro
Introduction	21	(1)
Outline of the method	22	(1)
Formaldehyde cross-linking in staged Drosophila embryos	23	(5)
Preparation of fly cages and collection of staged embryos	23	(1)
Optimizing cross-linking conditions	24	(4)
Immunoprecipitation of cross-linked embryonic chromatin and PCR amplification of the immunoprecipitated DNA	28	(3)
Analysing the enrichment of putative target sequences in the PCR-amplified DNA	31	(5)
Slot-blot analysis of the enrichment of putative PC target sequences	31	(1)
Mapping DNA target sites for Polycomb and GAGA factor in the Drosophila bithorax complex	31	(5)
Concluding remarks	36	(3)
References	37	(2)
Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP)	39	(20)
Karl Ekwall
Janet F. Partridge
Introduction	39	(1)
Fluorescence in situ hybridization (FISH) analysis of fission yeast	40	(8)
Preparation of probes	40	(3)
Cell fixation and cell-wall digestion	43	(5)
Chromatin immunoprecipitation from fission yeast	48	(11)
Fixation of yeast cells to maintain protein localization	49	(1)
Preparation of chromatin extract	50	(1)
Immunoprecipitation of chromatin	51	(4)
Analysis of immunoprecipitated DNA sequences	55	(1)
Acknowledgements	56	(1)
References	56	(3)
Isolation of vertebrate metaphase chromosomes and their analysis by FISH	59	(22)
Jeff Craig
Introduction	59	(1)
General equipment required for FISH	59	(1)
Production of metaphase chromosomes as substrates for FISH	60	(5)
Production of fixed metaphase chromosome spreads	60	(2)
Production of long prometaphase chromosomes	62	(1)
Isolation of suspensions of unfixed metaphase chromosomes	63	(2)
Spreading fixed chromosomes	65	(1)
Pretreatments of slides	66	(3)
Pretreatment of mitotic chromosome spreads	66	(1)
Salt extraction of isolated metaphase chromosomes	67	(2)
Labelling DNA probes	69	(3)
Choice of label	69	(1)
Nick translation	69	(1)
Random priming	70	(1)
Labelling by PCR	70	(1)
Quantifying label incorporation	71	(1)
Hybridization	72	(3)
Preparation of probes and slides	72	(1)
Hybridization	73	(2)
Detecting hybridized probe	75	(2)
Counterstaining and mounting	77	(4)
Simple counterstaining	77	(1)
Chromosome banding	77	(1)
Acknowledgements	78	(1)
References	78	(3)
Studying progression of vertebrate chromosomes through mitosis by immunofluorescence and FISH	81	(22)
Beth A. Sullivan
Peter E. Warburton
Introduction	81	(1)
Fundamental aspects of mitosis	81	(2)
The mitotic spindle	81	(1)
Chromosomes	82	(1)
Detecting centromere/kinetochore proteins on metaphase chromosomes	83	(4)
In situ hybridization following immunofluorescence	87	(3)
The use of anti-mitotic drugs	90	(2)
Colcemid/colchicine	90	(1)
Nocodazole	91	(1)
Vinblastine and other drugs	91	(1)
Cytochalasin/dihydrocytochalasin B (DCB)	91	(1)
Immunofluorescence on anaphase and telophase cells/chromosomes	92	(11)
C-anaphase: sister chromatid separation and anaphase in the presence of Colcemid	92	(2)
Anaphase chromosomes visualized on the mitotic spindle	94	(2)
Anaphase studies on cytokinesis-blocked cells	96	(4)
References	100	(3)
Analysis of mammalian interphase chromosomes by FISH and immunofluorescence	103	(22)
Joanna M. Bridger
Peter Lichter
Introduction	103	(2)
Preparation of sample material	105	(6)
Adherent cells	105	(1)
Suspension cells	106	(1)
DNA halo preparations	106	(1)
Fixation and permeabilization	107	(2)
Improving probe penetration	109	(2)
Probes	111	(2)
Chromosomal painting probes	111	(1)
Probe labelling	112	(1)
Fluorescence in situ hybridization	113	(3)
Denaturation	113	(2)
Hybridization	115	(1)
Washing	115	(1)
Detection of reporter molecules	116	(1)
Immunofluorescence in combination with FISH	116	(3)
Primary and secondary antibody incubations after FISH	118	(1)
Primary antibody incubation predenaturation and secondary antibody incubation postdenaturation	119	(1)
Primary and secondary antibody incubations prior to FISH denaturation	119	(1)
Reporter-conjugated primary antibody incubated predenaturation	119	(1)
Mounting the slides	119	(1)
Analysis	120	(5)
References	121	(4)
Fluorescence in situ hybridization in whole-mount tissues	125	(22)
Abby F. Dernburg
Introduction	125	(2)
Probe synthesis and labelling	127	(6)
General considerations	127	(1)
Why use fluorescece-based detection?	127	(1)
Choice of labelling and detection reagents	128	(3)
Probe synthesis	131	(2)
Fixation methods for whole-mount FISH	133	(3)
Hybridization methods	136	(5)
Troubleshooting	141	(2)
Microscopy and image analysis	143	(1)
Future directions	144	(3)
References	144	(1)
Acknowledgement	144	(3)
Analysing the substructure of mammalian nuclei, in vitro	147	(20)
Dean A. Jackson
Introduction	147	(1)
The nuclear matrix and nucleoskeleton	147	(1)
Methods used to analyse nuclear organization	148	(5)
The nuclear matrix	148	(1)
Nucleoids	149	(1)
The nuclear scaffold	150	(1)
The `low-salt' nuclear matrix	151	(1)
The nucleoskeleton	151	(2)
Studying the chromatin loops of different nuclear derivatives	153	(6)
Chromatin loops after hypertonic or hypotonic treatment	153	(1)
Chromatin loops under `physiological' conditions	154	(2)
Technical tips on cutting and electroeluting chromatin	156	(1)
The frequency and nature of attachment sites in different nuclear derivatives	157	(2)
The morphology of different nuclear derivatives	159	(1)
Assaying nuclear function and nuclear proteins in permeabilized cells	160	(4)
Labelling sites of replication and transcription in vitro	160	(1)
Technical tips on labelling sites of replication and transcription	161	(1)
Studying protein distribution relative to sites of transcription or replication in permeabilized cells	162	(2)
A typical example	164	(1)
Conclusions	164	(3)
References	165	(2)
Chromosome assembly in vitro using Xenopus egg extracts	167	(16)
Jason R. Swedlow
Introduction	167	(1)
Chromosome sttucture and biochemistry	167	(1)
Preparation of Xenopus egg extracts for chromatin and chromosome assembly in vitro	168	(7)
Xenopus egg maturation	168	(1)
Xenopus egg extracts	168	(1)
Chromosome assembly extracts---technical tips	169	(6)
Chromatin and chromosome assembly in vitro	175	(3)
Assembly and isolation of chromatin and chromosomes---technical tips	175	(3)
Immunofluorescence of in vitro assembled chromosomes	178	(2)
Functional analysis of the role of specific proteins in chromatin and chromosome structure by immunodepletion	180	(3)
Acknowledgements	181	(1)
References	182	(1)
Chromosome fragmentation in vertebrate cell lines	183	(16)
Christine J. Farr
Introduction	183	(1)
Telomere-associated chromosome fragmentation	183	(2)
Experimentally induced de novo telomere formation	185	(6)
Design of the telomere-seeding construct	185	(1)
Transfection of the telomere-seeding construct	186	(2)
Screening stable transfectants for de novo telomere formation	188	(3)
Targeted de novo telomere formation	191	(1)
Targeted truncation events in the recombination-proficient avian cell line DT40	192	(1)
The characterization of chromosomes modified by de novo telomere formation and fragmentation	193	(4)
Estimation of minichromosome size	194	(2)
Assays for mitotic stability of minichromosomes	196	(1)
Concluding remarks	197	(2)
References	197	(2)
List of suppliers	199	(8)
Index	207