Analytical Ultracentrifugation and Agarose Gel Electrophoresis as Tools for Studying Chromatin Folding in Solution (original) (raw)

Electrophoresis of Chromatin on Nondenaturing Agarose Gels Containing Mg[IMAGE]

Journal of Biological Chemistry, 1995

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg 2؉ , chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg 2؉ . In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg 2؉ and form cylindrical structures with a diameter of ϳ33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.

Fractionation of unfixed chromatin by buoyant-density centrifugation

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974

Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. The chromatin--DNA of minimally sheared chromatin (750--1000 base-pairs) bands as a single, sharp peak, almost completely separated from the chromatin ribonucleoprotein particles. More extensive shearing (to about 360 base-pairs) leads to a bimodal distribution of the chromatin--DNA in the gradients; all of the DNA is complexed with proteins.

Influence of Cell Fixation on Chromatin Topography

Analytical Biochemistry, 2000

Using in situ hybridization techniques, a fixation step must precede denaturation to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensional or 3D fixation and analysis) has become possible with the development of confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and analysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and analyses using different cell lines and different DNA probes. Fixation of cells by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear diameter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the outer parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolute centromere-membrane distance constant. On the other hand, euchromatin located in the inner parts of the nuclear volume is not shifted outside proportionally to the increase of molecular dimensions; consequently, the relative distances for the center of nucleus to gene are smaller after methanol-acetic acid fixation. The limitations of the analysis of dehydrated preparations for practical use and in research are discussed.

Chromatin assembly in isolated mammalian nuclei

Nucleic Acids Research, 1978

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [ 3 H]thymidine and then incubated in the presence of [a-32 P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32 P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3 H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease or DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs), (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest, (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.

Ultrastructure of transcriptionally competent chromatin

Nucleic Acids Research, 1990

We have examined a salt-soluble, transcriptionally competent gene-enriched fraction of chicken erythrocyte chromatin and compared it to bulk chromatin using the unique microanalytical capabilities of Electron Spectroscopic Imaging (ESI). The saltsoluble fraction is enriched 48 fold in 03-globin gene sequences and is also enriched in histones that are post-synthetically modified, including acetylation and ubiquitination. Differences between the two fractions are also apparent in the distribution of the two major forms of nucleoprotein structures, including (1) particles which present a circular profile and possess protein and DNA content nearly identical to that of the canonical nucleosome and account for 89% of particles in the bulk fraction but account for only 66% of the particles in the competent fraction, and (2) u-shaped particles which possess about 20% less protein mass than particles of circular profile and are about 1Ox more prevalent in the transcriptionally competent fraction than in the bulk. Additionally, elongated particles with protein and DNA content similar to the u-shaped objects are also seen in the competent fraction.

Partial Purification of the Template-Active Fraction of Chromatin: A Preliminary Report

Proceedings of the National Academy of Sciences, 1974

A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl 2 . The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA·RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.

Condensation of Interphase Chromatin in Nuclei of Synchronized Chinese Hamster Ovary (CHO-K1) Cells

DNA and Cell Biology, 2005

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.

Immunofluorescent study of chromatin proteins in cultured fibroblasts

Experimental Cell Research, 1974

Antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts were prepared by immunization of rabbits. Immunofluorescent studies showed species-specificity of these antichromatin antibodies. Furthermore, using anti-3T6 chromatin antibodies against 3T6 cells and anti-WI-38 chromatin antibodies against WI-38 cells, we observed, by immunofluorescent techniques, granular fluorescence i&the diCfusely stained nucleus and diffuse fluorescence in the cytoplasm. These results raise the possibility of the presence of a cytoplasmic pool of chromatin proteins.