Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.     

Preparation of megabase-size DNA from plant nuclei

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ . This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.     

Multiple DNA polymerase activity solubilized from higher plant chromatin

DNA polymerase preparations solubilized from chromatin of unwashed and washed sugar beet storage tissue exhibit multiple activity peaks when separated by DEAE-Sephadex chromatography. Activity peaks isolated from unwashed and washed tissue required Mg²⁺, all four deoxynucleoside triphosphates and added DNA. Activity from peaks isolated from washed tissue was completely sensitive to pancreatic DNase and insensitive to RNase. Enzyme activity was increased in the tissue during the washing period. In addition, there was a drastic change in template specificity from single-stranded primer in unwashed tissue to double-stranded DNA in washed tissue.     

Cryo-electron microscopy reveals high-resolution structures of 30-nm chromatin fibers in vitro

<正>Human chromosomes are the largest and most visible structures involved in the transfer of genetic information.The most striking property of a chromosome is that about three billion base pairs of the human genome are compacted into a nucleus of only several micrometers in diameter.It is well known that DNA in eukaryotic cells is initially packaged into nucleosomes by means of extensive association     

Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei

The morphological effects of epidermal growth factor (EGF) on human carcinoma cells A-431 have been examined by scanning electron microscopy. These flat polygonal cells normally exhibit only small membrane folds, but show extensive ruffling and extension of filopodia within 5 min of exposure to EGF at 37 degrees C. This ruffling activity is transient, subsiding within another 5--15 min, but several other changes in surface morphology follow. Within the first hour of exposure to the hormone, the cell surface becomes exceedingly smooth and the nuclei seem to protrude above the plane of the otherwise thin monolayer, giving the cells a "fried egg" appearance. Cells at the edges of colonies gradually retract from the substrate, leading to reorganization, by 12 h, of the monolayer into multilayered colonies. EGF thus induces both rapid and long-term alterations in the morphology of these epidermoid cells.     

Supranucleosomal organization of chromatin fibers in nuclei of Drosophila S2 cells

Earlier, the interphase chromatin structures could not be visualized due to the stickiness of the nuclear material. We have reduced stickiness by the reversal of permeabilization allowing the isolation and microscopic imaging of interphase chromatin structures. By using a high resolution of synchronization, collecting 36 elutriation fractions, we show that major intermediates of chromatin condensation include: (a) decondensed veillike chromatin at the unset of the S phase (2.0-2.2 C-value), (b) polarization of veiled chromatin (2.2-2.6 C), (c) fibrous chromatin (2.6-3.0 C), chromatin bodies (3.0-3.3 C), early precondensed chromosomes (3.3-3.6). The compaction of Drosophila chromosomes did not reach that of the mammalian cells in the final stage of condensation (3.6-4.0 C). Drosophila chromosomes consist of smaller units called rodlets. Results demonstrate that nucleosomal chromatin (beads on string) does not form a solenoid structure; rather, the topological arrangement consists of meandering and plectonemic loops.     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.

BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

Spontaneous access to DNA target sites in folded chromatin fibers

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from ∼ 3-fold decreases to ∼ 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as ∼ 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.     

Satellite DNA content of chromatin fractions isolated from Eco R1-digested mouse liver nuclei

Two chromatin fractions have been prepared from Eco R1-digested mouse liver nuclei. One of these, containing 7–10% of the total chromatin DNA, contains no satellite DNA. The other, constituting 9% of the total chromatin DNA, is four- to five-fold enriched in satellite DNA.     

Isolation of nuclei for chromatin analysis in fission yeast.

The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.