Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome
TL;DR: All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromaatin and with the isoform.
Abstract: The pattern of histone H4 acetylation in different genomic regions has been investigated by immunoprecipitating oligonucleosomes from a human lymphoblastoid cell line with antibodies to H4 acetylated at lysines 5, 8, 12 or 16. DNA from antibody-bound or unbound chromatin was assayed by slot blotting. Pol I and pol II transcribed genes located in euchromatin were shown to have levels of H4 acetylation at lysines 5, 8 and 12 equivalent to those in input chromatin, but to be slightly enriched in H4 acetylated at lysine 16. In no case did the acetylation level correlate with actual or potential transcriptional activity. All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromatin and with the isoform. Two single copy genes that map within or adjacent to blocks of paracentric heterochromatin are depleted in H4 acetylated at lysines 5, 8 and 12, but not 16. Consensus sequences of repetitive elements of the Alu family (SINES, enriched in R bands) were associated with H4 that was more highly acetylated at all four lysines than input chromatin, while H4 associated with Kpn I elements (LINES, enriched in G bands) was significantly underacetylated.
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15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
01 Jan 1979
TL;DR: The Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro, and Generalized Biological Effects.
Abstract: of Part A.- Section I: What is the Chromatin?.- Properties and Composition of Isolated Chromatin.- Expressed and Nonexpressed Portions of the Genome: Their Separation and Their Characterization.- Discussion.- Section II: Physical, Chemical and Biological Techniques for Studying Nucleosome, Chromatin, Chromosome and Nuclei.- Electron Microscopy: A Tool for Visualizing Chromatin.- Transcriptional Control of Native Chromatin.- Circular Dichroism of DNA, Protein and Chromatin.- Important Hydrodynamic and Spectroscopic Techniques in the Field of Chromatin Structure.- Preparation and Analysis of Core Particles and Nucleosomes: A Conveinient Method For Studying the Protein Composition of Nucleosomes Using Protamine-Release into Triton-Acid-Urea Gels.- The Interaction of Histones with DNA: Equilibrium Binding Studies.- Nucleosome Shape and Structure in Solution from Flow Birefringence.- Scattering and Diffraction by Neutrons and X-rays in the Study of Chromatin.- Nuclear Magnetic Resonance Studies of Nucleic Acids and Proteins.- Techniques for Cytochemical Studies of the Nucleus and its Substructures.- Chromatin Study in Situ: I. Image Analysis.- Chromatin Study in Situ: II. Static and Flow Microfluorimetry.- Chromatin Study in Situ: III. Differential Effects of Feulgen Hydrolysis.- Scanning and Flow Photometry of Chromosomes.- Discussion.- Section III: Various Levels of Chromatin Organization and Mechanisms for Transcriptional Control.- Histones Assembly and Their Structural Role for Nucleosome Core.- Nuclease Digestion and the Structure of Chromatin.- Reconstitution of Nucleosomes.- Conformation of Polynucleosomes in Low Ionic Strength Solution.- Chromatin Structure: Relation of Nucleosomes of DNA Sequences.- Histone Complexes, Nucleosomes, Chromatin and Cell-Cycle Dependent Modification of Histones.- Evidence for Superstructures of Wet Chromatin.- Chromatin Fractionation and the Properties of Transcriptionally Active Regions of Chromatin.- Chromatin Reconstitution and Non-Histone Proteins.- Discussion.- Section IV: Structure-Function of the Genetic Apparatus and Cell Cycle, Aging, Neoplastic Transformation, Differentiation, Chemical Carcinogenesis.- The Structure and Function of Chromatin in Lower Eukaryotes.- Chromatin Structure from Angstrom to Micorn Levels, and Its Relationship to Mammalian Cell Proliferation.- Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro.- Neoplastic Transformation: The Relevance of in Vitro Studies for the Understanding of Tumor Pathenogenesis and Neoplastic Growth.- Cell Differentiation and Malignancy in Leukemia.- Cellular Morphometry in Transformation, Differentiation and Aging.- Basic Mechanisms in Chemical Carcinogenesis.- Carcinogen Induced Alteration in Gene Packing and Its Possible Significance in Carcinogenesis.- Covalent Binding of a Carcinogen to DNA as a Probe of Chromatin Structure.- Carcinogenesis, DNA Repair and Chromatin.- Electromagnetic Induction of Electrochemical Information at Cell Surfaces: Application to Chromatin Structure Modification.- Discussion.- Section V: Review and Summary of the Genetic Apparatus.- Session I: Basic Components of the Genetic Apparatus.- Session II: The Second Level of Organization - Chromatin.- Session III: The Third Level of Organization.- Session IV: Generalized Biological Effects.
1,058 citations
Book•
01 Jan 1987
TL;DR: The induction and enumeration of antibody-forming cells in vitro and the development of human B lymphoblastoid cell lines using epstein are studied.
Abstract: Preparation of lymphocytes and accessory cells Preparation of lymphocyte subpopulations Fractionation of lymphocytes by immunomagnetic beads Immunofluorescence and immunohisto-chemistry The induction and enumeration of antibody-forming cells in vitro In vitro culture of T cell lines and clones Generation of human B lymphoblastoid cell lines using epstein Limiting dilution analysis Lymphocyte proliferation assays Assays for interleukins and other related factors Biochemical characterization of lymphocyte surface antigens
185 citations
TL;DR: Clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligon nucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences is obtained.
Abstract: Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dU
100 citations
Book•
01 Jan 2000
TL;DR: The objective is to establish a protocol for quantification of antigen-specific T-cells HLA -peptide tetrameric complexes and investigate the role of T-cell reprograming in the selection of lymphocytes for HLA typing.
Abstract: Preface Preparation of lymphocytes and idenfication of lymphocyte subpopulations Immunohistochemistry of lymphoid organs T and B-cell hybridomas Murine T-cell culture Human CD4+ T-cell culture Human Cytotoxic T-cell culture Limiting dilution analysis for quantification of antigen-specific T-cells HLA -peptide tetrameric complexes Expansion of human T-cells for immunotheraphy HLA typing Characterisation of lymphocyte surface markers Apoptosis assays for lymphocytes Thymic organ culture Index
29 citations