Drosophila nucleosomes contain an unusual histone-like protein.

Palmer, Douglas K.; Snyder, L. A.; Blumenfeld, Martin

doi:10.1073/pnas.77.5.2671

Cited by 28 publications

(24 citation statements)

References 30 publications

(16 reference statements)

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“…All operations were performed at 4°C unless otherwise indicated. Nuclei were prepared from adult rat livers, using slight modifications of a previously published procedure (28). Freshly excised, minced livers were homogenized in 8 to 10 volumes of buffer A', using a Potter-Elvehjem apparatus.…”

Section: Methodsmentioning

confidence: 99%

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

Palmer¹,

Margolis²

1985

Mol. Cell. Biol.

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We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements. Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low-and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei. The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy. Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size. The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin. These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone HI. Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.

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Section: Methodsmentioning

confidence: 99%

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

Palmer¹,

Margolis²

1985

Mol. Cell. Biol.

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“…An H2A.F/Z variant has been identified in a wide variety of eukaryotes including Tetrahymena (hv1), budding yeast (HTA3 and HTZ1), Drosophila (D2, H2A.2, H2AvD, His2AvD, His2Av, and H2Av), chickens (H2A.F), mice (H2A.Z), and humans (H2A.Z) suggesting that incorporation of H2A.F/Z into chromatin is evolutionarily ancient and conserved. H2A.F/Z typically constitutes 5-10% of total H2A proteins in the chromatin of cells (18,19). H2A.F/Z is essential in Tetrahymena, Drosophila, and mice, but its essential function is not known (20 -22).…”

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confidence: 99%

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

Leach

Mazzeo

Chotkowski

et al. 2000

Journal of Biological Chemistry

123

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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“…It was discovered by Alfageme et al (8) and designated D2, or "Drosophila 2." D2 is nucleosomal and H2A-like in its amino acid composition (9). In this article, we establish that it functions as an H2A during the in vitro reconstitution of Drosophila histone octamers and thus is an H2A histone.…”

mentioning

confidence: 90%

“…We therefore rename D2 as H2A.2 and the major H2A histone as H2A. 1. H2A.2 occurs with a frequency of approximately one molecule per five nucleosomes in 0-to 18-hr Drosophila embryos, adult heads, and SL2 cells (9,10). It has been conserved during the evolution of Drosophila.…”

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confidence: 99%

Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes

Donahue¹

1986

Proc. Natl. Acad. Sci. U.S.A.

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Drosophila chromatin contains two antigenically distinct H2A histones, H2A.1 and H2A.2. Indirect immunofluorescence analyses revealed that anti-H2A.1 binding was distributed throughout polytene chromosomes, whereas anti-H2A.2 binding was Interband-specific. Thus, H2A.2 probably contributes to the less compacted structure of interbands. Since each band-interband region is thought to contain a single gene, our results suggest that the distribution of H2A.2 echoes the functional organization of the Drosophila genome. Similar H2A histones occur in eukaryotes ranging from protozoa to mammals. Their placement might be an important determinant of chromatin structure.Eukaryotic DNA is compacted into nucleosomes by octamers containing two molecules of each of the four histone types: H2A, H2B, H3, and H4 (reviewed in refs. 1 and 2). Primary sequence histone variants can generate nucleosome diversity. In the sea urchin, sperm-specific histones are replaced by maternal cleavage-stage histones during male pronuclear decondensation (3). In the developing embryos, stage-specific histones (4) affect nucleosomal structure and stability (5). In Tetrahymena, an H2A-like histone, hvl, occurs in the transcriptionally active macronucleus but not in the transcriptionally inactive micronucleus (6). The appearance of hvl in the developing macronucleus coincides with the onset of RNA synthesis (7), suggesting a relationship between gene activity and the distribution of specific histones.In Drosophila melanogaster, only one histone sequence variant has been reported. It was discovered by Alfageme et al. (8) (9), 2 mg of poly(glutamic acid) (Sigma, type III-B) per ml, 10 mM triethanolamine-HCl (pH 8.0), 0.1 M NaCl, and 10 mM 2-mercaptoethanol (17). They were shaken gently (12-14 hr; 20-22°C), diluted 1:10 with 55 mM Na2B407 (pH 9.5), combined with 1/50th vol of dimethyl suberimidate (Sigma; 50 mg/ml in dimethyl sulfoxide) at four 15-min intervals, incubated (15 min), precipitated with 20% trichloroacetic acid (0WC), washed (acetone/0.2% HCl and acetone), and air dried. In some experiments, histone octamers were crosslinked reversibly with dithiobis(succinimidylpropionate) (Lomant's reagent; ref. 18). In these cases, 2-mercaptoethanol was omitted from the reconstitution mixture.Crosslinked histone complexes were separated electrophoretically on 10 x 0.1 cm 5% acrylamide slab gels containing 0.1% NaDodSO4 and 0.1 M sodium phosphate (pH 7.1) (19), stained with Coomassie brilliant blue R, and destained. Bands containing reversibly crosslinked histone octamers, produced with Lomant's reagent, were excised, incubated in 62.5 mM Tris, pH 6.8/5% glycerol/10 mM 2-mercaptoethanol (60 min; 22-23°C), and electrophoresed in 18% acrylamide/NaDodSO4 gels (9).Preparation of Antibodies. H2A.2 was purified from total H2A (9) by electrophoresis in discontinuous gels containing 0.5% Triton X-100, 6% HOAc, and 8 M urea (20), excised from the gels, electrophoresed in 15% acrylamide/NaDod-S04 gels (9), excised, electrophoresed into Tris/glycine tray buff...

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Drosophila nucleosomes contain an unusual histone-like protein.

Cited by 28 publications

References 30 publications

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

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

Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes

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