Immunological Probes for Chromatin Structure

Bustin, Michael; Kurth, Paul D.; Moudrianakis, Evangelos N.; Goldblatt, Drora; Sperling, Ruth; Rizzo, William B.

doi:10.1101/sqb.1978.042.01.039

Cited by 17 publications

(8 citation statements)

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“…When acid-squashed chromosomes are employed to map the distribution of histone proteins, the results obtained are dependent on details of the chromosome isolation and fixation procedures. For example, there is a marked but variable lowering of anti-HI-induced immunofluorescence especially over puffs (Bustin et al 1978). However, when chromosomes isolated at neutral pH are treated with monoclonal antibodies against histone HI, the marked tendency to lowered fluorescence over puffs is not apparent (Fig.…”

Section: Nucleosome Module Present In Spreads Of Native Salivary Chromentioning

confidence: 97%

“…4c). It is likely that acetic acid extracts histone HI from salivary chromosomes and that this extraction may be only partially prevented by formaldehyde fixation (Bustin et al 1978). Thus it would appear that the more rigorous approach of completely avoiding exposure of the chromosomes to acid offers definite advantages for in situ protein localization analyses.…”

Section: Nucleosome Module Present In Spreads Of Native Salivary Chromentioning

confidence: 99%

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Native Salivary Chromosomes of Drosophila melanogaster: Retrospect and Prospect

Hill¹

1985

Aust. Jnl. Of Bio. Sci.

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A method has been developed which, for the first time, allows the preparation of mappable cytological spreads of salivary chromosomes from D. melanogaster without exposure to acid fixatives. These isolated native chromosomes show the best preservation of ultrastructure observed to date-ribonucleoprotein particles may be seen to be organized in linear arrays in transcriptionally active puffs and the repeating nucleosome module is present. Native salivary chromosomes are proving useful for the localization of nuclear proteins both at the light microscope and ultrastructural levels. They display only background-level binding of antibodies specific for the Z-DNA conformation. However, Z-DNA immunoreactivity is activated by exposure to acid fixative, first in interbands and then in bands. The Z-conformation in the chromosomes is held in place by elastic torsional strain which appears in the DNA following acid fixation. Native D. melanogaster salivary chromosomes offer promise for enabling the probing of the chromatin of known genetic loci for properties dependent on the preservation of macromolecular integrity.

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Section: Nucleosome Module Present In Spreads Of Native Salivary Chromentioning

confidence: 97%

Section: Nucleosome Module Present In Spreads Of Native Salivary Chromentioning

confidence: 99%

Native Salivary Chromosomes of Drosophila melanogaster: Retrospect and Prospect

Hill¹

1985

Aust. Jnl. Of Bio. Sci.

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“…In contrast, the immunoelectron microscopic technique requires clear visualization of the antibody bound to the nucleosome. Often the antibody "hugs" the nucleosome very tightly and the Fc portion barely protrudes from the periphery of the nucleosome (9). Only those nucleosomes in which the diameter increased by at least 50% were scored as positives for antibody binding.…”

Section: Nucleosome Heterogeneimmentioning

confidence: 99%

“…The presence of histones on the transcriptionally active fiber was visualized by immunoelectron microscopy (9). The specific binding of affinity-purified antihistone antibodies to spreads of chromatin fibers prepared from Drosophila embryos was demonstrated by using ferritinconjugated antibodies.…”

Section: Transcriptionally Active Chromatinmentioning

confidence: 99%

“…These results suggest that the protein may be involved in modulating the structure of selected regions in the chromosome. In transcriptionally active puffs the antigenic determinants of The presence of histones on the transcriptionally active fiber was visualized by immunoelectron microscopy (9). The specific binding of affinity-purified antihistone antibodies to spreads of chromatin fibers prepared from Drosophila embryos was demonstrated by using ferritinconjugated antibodies.…”

Section: Transcriptionally Active Chromatinmentioning

confidence: 99%

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Immunochemical analysis of the structure and function of chromosomal proteins

Bustin

1987

Cytometry

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Immunochemical approaches are useful in studying the nuclear organization and cellular function of chromosomal components. Antibodies specific to histones and to defined nonhistone proteins have been used to study nucleosome heterogeneity, to visualize the The structure of chromatin and chromosomes and the regulation of the genetic information encoded in DNA is largely dependent on a variety of interactions between the nucleic acid and protein components of the genome. In both interphase chromatin and metaphase chromosomes, the nucleoprotein fiber is built from a n array of repeating subunits, known as core particles, which contain about 146 base pairs of DNA complexed with two molecules of each histone H2A, H2B, H3, and H4. Adjacent core particles are covalently linked through a DNA segment, resulting in a fiberlike structure resembling a beaded string. The beaded chromatin fiber is further packed to yield the observed structure of chromatin and chromosomes (for reviews on chromatin structure see 15,18,26).Histones are the predominant class of nuclear proteins. Another class of nuclear proteins, collectively known as the nonhistone chromosomal proteins, is composed of a great number of molecular species. Members of this class of proteins are involved in various regulatory and structural aspects of the chromatin fiber. The chromatin fiber is a dynamic structure: gross morphological changes in the superstructure of the fiber, which occur during the cell cycle, are easily observable by light microscopy. The chromatin fiber is the substrate for the complex processes of transcription, replication, and repair. These metabolic processes often are accompanied by changes in the nonhistone protein constituents of the fiber and involve structural reorganization within the nucleosome. Furthermore, both the protein and the nucleic acid components of the chromatin fiber are subject to degradation, modification, and repair-processes which may alter the manner in which they interact with each other. Obviously, structural studies of the chromatin fiber have to take into account the dynamic properties of the complex and require techniques which can presence of histone in transcriptionally active chromatin, and to isolate DNA sequences associated with specific chromosomal proteins.Key terms: Histone, nonhistone, chromatin, an tibodies provide information on the native, in situ, organization of defined chromosomal components at various stages of chromatin organization.Because serological reactions occur under conditions which do not alter markedly the structure of chromatin and chromosomes, and since antibodies can react specifically with a n antigen under a variety of conditions, immunochemical approaches are uniquely suited for a variety of studies on the organization of nuclear proteins in the nucleoprotein fiber. Quantitative immunocytological techniques can be used to obtain information on local variations in antigen concentrations in metaphase and polytene chromsomes and in individual cell nuclei (1). Research involving immu...

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Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry

et al. 1984

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A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.

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Immunological Probes for Chromatin Structure

Cited by 17 publications

References 0 publications

Native Salivary Chromosomes of Drosophila melanogaster: Retrospect and Prospect

Native Salivary Chromosomes of Drosophila melanogaster: Retrospect and Prospect

Immunochemical analysis of the structure and function of chromosomal proteins

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry

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