9780199636983

Chromosome Structural Analysis A Practical Approach

by
  • ISBN13:

    9780199636983

  • ISBN10:

    0199636982

  • Format: Paperback
  • Copyright: 1999-07-29
  • Publisher: Oxford University Press

Note: Supplemental materials are not guaranteed with Rental or Used book purchases.

Purchase Benefits

  • Free Shipping On Orders Over $35!
    Your order must be $35 or more to qualify for free economy shipping. Bulk sales, PO's, Marketplace items, eBooks and apparel do not qualify for this offer.
  • Get Rewarded for Ordering Your Textbooks! Enroll Now
List Price: $165.00 Save up to $16.50
  • Rent Book $148.50
    Add to Cart Free Shipping

    TERM
    PRICE
    DUE

Supplemental Materials

What is included with this book?

  • The New copy of this book will include any supplemental materials advertised. Please check the title of the book to determine if it should include any access cards, study guides, lab manuals, CDs, etc.
  • The Rental copy of this book is not guaranteed to include any supplemental materials. Typically, only the book itself is included. This is true even if the title states it includes any access cards, study guides, lab manuals, CDs, etc.

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

Rewards Program

Write a Review