Subnuclear Components

1 Isolation of Animal Cell Nuclei

The Structure and Function of Nuclei

Nuclear Structure

Nuclear Function

Reasons for Isolating Nuclei

Problems Encountered when Isolating Nuclei

Cellular Heterogeneity

Cell Disruption

Isolation Media

Separation of Nuclei from the Brei

Criteria of Purity

Specific Methods for Different Tissues

Lower Eucaryotes

Invertebrates and Amphibia

Tissue-Culture Cells

Vertebrate Organs and Tissues

Tumor Tissues

Conclusions

References


2 Isolation of Nuclear Envelopes from Whole Tissue

The Nuclear Envelope In Situ

Morphology

Cytochemistry

Chemical Composition

General Consideration of Isolation Methods

Tonicity of Isolation Media

Ionic Composition of Isolation Media

pH of Isolation Media

Temperature

Homogenization Techniques

High-Density Sucrose Centrifugation

Isolation of Nuclear Envelopes from Nuclei

Nuclear Envelope Isolation Methods

Evaluation of Isolation Methods

Morphological Assessment

Composition

Enzyme Activities

Biochemical Assessment of Contamination

Conclusions

References


3 Isolation of Nucleoli

Isolation of Nucleoli from Oöcytes

Isolation of Nucleoli from Nuclei

Disrupting Nuclei by Deoxyribonuclease

Disrupting Nuclei by Conication

Disrupting Nuclei by Pressure Methods

Isolation of Nuclei and Nucleoli in Citric Acid

Isolation of Nucleoli by Other Methods

Isolation of Nucleoli from Physarum

Isolation of Plant Nucleoli

Conclusions

References


4 Preparation, Characterization and Fractionation of Chromatin

Preparation of Chromatin from Inter-phase Cells

From Animal Tissues and Cells

From Cells other than Animal Cells

Preparation of Chromatin from Meta-phase Cells

Isolation of Meta-phase Cells

Isolation, Purification and Fractionation of Meta-phase Chromosomes

Contaminants of Chromatin

Contaminants of Cytoplasmic Origin

Contaminants of Nuclear Origin

Characteristics of Isolated Chromatin

Chemical Composition

Physicochemical Properties

Template Activity

Electron Microscopic Studies

Immunological Properties

Fractionation of Chromatin

Features Distinguishing Active and Inactive Chromatin

Fragmentation of Chromatin

Characterization of Chromatin Fractions

Separation of Chromatin Fractions

Conclusions

Dissociation and Reassociation of Chromatin

Partial Deproteinization

Reconstitution

Conclusions

References


5 Fractionation and Isolation of Histones

Isolation and Purification of the Five Main Histone Fractions of Calf Thymus during One Preparation

Details of the Procedure

Comments on the Procedure

Purification of Histone Fractions

Gel Exclusion Chromatography for the Separation of All Five Histones

Preparation of Individual Histone Fractions

Preparation of Histone F1

Preparation of Histone Fractions other than F1

Preparation of the Chicken-Erythrocyte-Specific Histone F2C

Preparation of Whole Unfractionated Histones from Calf Thymus

Preparation of Histones from Tissues Other than Thymus

Characterization of Products

Storage of Histones

References


6 Preparation and Properties of Chromatin Non-Histone Proteins

Biological Relevance of Non-Histone Proteins

Control of Gene Expression

Structure of Chromatin

Post-Synthetic Modifications

Methods for Isolating Non-Histone Proteins

Methods Applied to Dehistoned Chromatins

Methods Applied to Dissociated Chromatins

Selective Extraction Procedures

Fractionation of Non-Histone Proteins

Ion-Exchange Chromatography

Iso-Electric Docusing

Gel Filtration

Characterization of Non-Histone Proteins

Electrophoresis

Amino Acid Composition

Immunospecificity

Factors Affecting the Composition of Non-Histone Proteins

Methodological Artifacts

Contamination by other Cell Constituents

Preparation of Nuclei and Chromatin

Methods Developed in the Author's Laboratory

Materials

Fractionation of Chromatin

One-Dimensional Gel Electrophoresis in Sodium Dodecyl Sulphate

Two-Dimensional Gel Electrophoresis

Fractionation of non-Histone Proteins on a Preparative Scale

Notes on the Methods

Conclusions

References


7 Isolation of Nuclear RNA

Problems Encountered in Isolating Nuclear RNA

Amounts

Contaminants

Degradation

Aggregation

Comparison of Methods

Method for Isolating Nuclear RNA

Details of Procedure

Extraction of Total Cellular RNA

Extraction of Nucleolar RNA

Extraction of Low-Molecular-Weight Nuclear RNA

Assessment of Nuclear RNA Preparations

Amounts Obtained

Contaminants

Degradation and Aggregation

Fractionation of Nuclear RNA

References


8 Isolation of DNA from Eucaryotic Cells

Properties of DNA and Problems Associated with its Isolation

How 'Big' is DNA?

To what Extent is the DNA Duplex Intact

Association of Protein and RNA with DNA

DeoxyriboTiuclease Activity in Ribonuclease and Proteolytic Enzymes

Criteria for the Selection of an Isolation Procedure

Comparison of Procedures Described

Methods of Assessing Size, Integrity and Purity of DNA

Estimation of Molecular Weight

Estimation of Integrity

Estimation of DNA and Contaminants

Isolation of DNA: Procedure I

Description of Procedure I

Analysis of Steps in Procedure I, and Possible Modifications

Properties of Product of Procedure I

Comments on Procedure I

Isolation of DNA: Procedure II

Description of Procedure II

Analysis of Steps in Procedure II

Properties of Product of Procedure II

Isolation of DNA: Procedure III

Description of Procedure III

Analysis of Steps in Procedure III, and Properties of the Product

General Conclusions

References

Index