Histone localization in polytene chromosomes by immunofluorescence.

Kurth, Paul D.; Moudrianakis, Evangelos N.; Bustin, Michael

doi:10.1083/jcb.78.3.910

Cited by 22 publications

(5 citation statements)

References 19 publications

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“…4b). This result agrees with earlier studies (37) and is consistent with a uniform distribution of H2A-containing nucleosomes along the chromosome paralleling the density of DNA. The H2A result contrasts markedly with the complex immunofluorescence pattern observed for H2Av, which did not correlate with the banded structure of the chromosome (compare Figs.…”

Section: Distribution Of H2av In Polytenesupporting

confidence: 82%

Histone H2A.Z Is Widely but Nonrandomly Distributed in Chromosomes of Drosophila melanogaster

Leach

Mazzeo

Chotkowski

et al. 2000

Journal of Biological Chemistry

124

107

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Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1.688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.The basic unit of chromatin in eukaryotes is the nucleosome. A nucleosome consists of 146 base pairs of DNA wrapped around an octamer of histone proteins H2A, H2B, H3, and H4 (1). Although chromatin is a highly reiterative structure, cells create heterogeneity in the structure of nucleosomes to facilitate and regulate DNA-mediated processes such as transcription. Heterogeneity is created, in part, by posttranslational modifications of histone proteins, including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation (2-4). Acetylation status of the amino termini of histones H3 and H4, in particular, plays a important role in transcriptional regulation (2, 5).Heterogeneity in nucleosome structure also results from incorporation of variant histone proteins into the nucleosome. In contrast to the canonical histones, which are multicopy genes expressed during S-phase of the cell cycle, variant histones are encoded by single copy genes that differ in amino acid sequence from S-phase histones and whose expression is not limited to S-phase (6, 7). Variant histones allow specialization of nucleosome structure for specific purposes. Sperm-specific variant histones, for example, facilitate the dramatic compaction of DNA that occurs during spermatogenesis (4,8). A specific variant of histone H3 is incorporated specifically at centromeres creating a specialized chromatin structure required for proper function of the kinetochore (9 -14), and macro-H2A, a histone H2A variant, localizes preferentially to the inactive X chromosome in mammals and may alter chromatin in a way that helps suppress transcription (15). These and other variants have been identified for histones H2A,...

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Section: Distribution Of H2av In Polytenesupporting

confidence: 82%

Histone H2A.Z Is Widely but Nonrandomly Distributed in Chromosomes of Drosophila melanogaster

Leach

Mazzeo

Chotkowski

et al. 2000

Journal of Biological Chemistry

124

107

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“…7 A) while tagged histone H3-HA1 localized throughout the nucleus as expected for histone H3 (Fig. 7 D; Kurth et al, 1978). The chimeric CA/H3-HA1, possessing the unique NH2 terminus of CENP-A, failed to localize at centromeres but rather was distributed throughout the nucleus (Fig.…”

Section: The Histone Fold Domain Of Cenp-a Is Required For Centromerimentioning

confidence: 62%

Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere.

Sullivan

Hechenberger

Masri

1994

The Journal of Cell Biology

427

292

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Abstract. Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of eentromeric chromatin, we have cloned a human eDNA that encodes the 17-kD historic-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.

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“…In some cases the transcriptionally active loci can be detected as "puffs" by phase-contrast microsopy or, more reliable, by autoradiographic techniques using labeled uridine. Immunofluorescence studies with antihistone sera produce a distinct banding pattern reminiscent of that observed by phase-contrast microscopy (22) (Fig. 3).…”

Section: Transcriptionally Active Chromatinmentioning

confidence: 52%

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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Histone localization in polytene chromosomes by immunofluorescence.

Cited by 22 publications

References 19 publications

Histone H2A.Z Is Widely but Nonrandomly Distributed in Chromosomes of Drosophila melanogaster

Histone H2A.Z Is Widely but Nonrandomly Distributed in Chromosomes of Drosophila melanogaster

Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere.

Immunochemical analysis of the structure and function of chromosomal proteins

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