Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers

1998 ◽  
Vol 111 (12) ◽  
pp. 1707-1715
Author(s):  
A. Bermudez ◽  
S. Bartolome ◽  
J.R. Daban
Keyword(s):  
Radial Distribution ◽  
Direct Evidence ◽  
Dna Loops ◽  
Entry Points ◽  
Partial Denaturation ◽  
Chromatin Fibers ◽  
High Concentrations ◽  
Chicken Erythrocytes ◽  
Top View ◽  
Electrostatic Repulsions

To examine the internal structure of chromatin fibers, we have developed procedures for partial denaturation of small chromatin fragments (8–30 nucleosomes) from chicken erythrocytes. Electron micrographs of samples prepared under conditions that cause nucleosome dissociation show rods and loops projecting from short compact fibers fixed by glutaraldehyde in 1.7 mM Mg2+. According to previous studies in our laboratory, these images correspond to the top view of partially denatured fibers. Our results indicate that rods and loops consist of extended duplex DNA of different lengths. DNA in loops is nicked, as demonstrated by experiments performed in the presence of high concentrations of ethidium bromide. Length measurements indicate that the radial projections of DNA are produced by unfolding of nucleosomal units. Loops are formed by DNA from denatured nucleosomes in internal positions of the fiber; DNA from denatured nucleosomes in terminal positions form rods. Our micrographs show clearly a radial distribution of DNA loops and rods projecting from fibers. Rods are orthogonal to the surface of the chromatin fragments. Considering that the high ionic strength used in this study (0.8-2.0 M NaCl) neutralizes the electrostatic repulsions between rods and fiber, this observation suggests that rods are extensions of nucleosomes radially organized inside the fiber. The position of the entry points of DNA loops into the fiber could be influenced by constraint on loops, but our results showing that the arc that separates these points in dinucleosome loops is relatively short suggest that consecutive nucleosomes are relatively close to each other in the folded fiber.

scholarly journals Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length.

1994 ◽  
Vol 125 (1) ◽  
pp. 11-19 ◽  
Author(s):  
C L Woodcock
Keyword(s):  
Cell Types ◽  
Repeat Length ◽  
Strong Positive Correlation ◽  
Whole Cells ◽  
Chromosome Architecture ◽  
Chromatin Fibers ◽  
The Moment ◽  
Chicken Erythrocytes ◽  
Patiria Miniata

Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20-bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo-immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been established. The demonstration of chromatin fibers in unfixed whole cells establishes an unequivocal baseline for the study of native chromatin and chromosome architecture. The significant differences between chromatin fibers in nucleo and after isolation supports a previous observation (P. J. Giannasca, R. A. Horowitz, and C. L. Woodcock. 1993. J. Cell Sci. 105:551-561), and suggests that structural studies on isolated material should be interpreted with caution until the changes that accompany chromatin isolation are understood.

scholarly journals The human complement system: assembly of the classical pathway C3 convertase

1980 ◽  
Vol 189 (1) ◽  
pp. 173-181 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Direct Evidence ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Classical Pathway ◽  
Human Complement ◽  
Stabilizing Effect ◽  
Excess Substrate ◽  
High Concentrations ◽  
Low Ionic Strength

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

scholarly journals Human cohesin compacts DNA by loop extrusion

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1345-1349 ◽  
Author(s):  
Yoori Kim ◽  
Zhubing Shi ◽  
Hongshan Zhang ◽  
Ilya J. Finkelstein ◽  
Hongtao Yu
Keyword(s):  
Adenosine Triphosphate ◽  
Single Molecule ◽  
Adenosine Triphosphatase ◽  
Direct Evidence ◽  
Atp Hydrolysis ◽  
Average Rate ◽  
Molecular Machine ◽  
Single Molecule Imaging ◽  
Dna Loops ◽  
Dna Compaction

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

Internal structure of the 30 nm chromatin fiber

1994 ◽  
Vol 107 (11) ◽  
pp. 2983-2992 ◽  
Author(s):  
S. Bartolome ◽  
A. Bermudez ◽  
J.R. Daban
Keyword(s):  
Electron Microscope ◽  
Central Region ◽  
Chromatin Fiber ◽  
Central Hole ◽  
Structural Elements ◽  
Structural Components ◽  
Microscope Images ◽  
Annular Zone ◽  
Chicken Erythrocytes ◽  
Top View

In the presence of 1.7 mM Mg2+, the diameter of the circular structures produced by small chromatin fragments isolated from chicken erythrocytes remains essentially unchanged when the number of nucleosomes in these fragments increases from 10 to 36. In contrast, the results obtained in unidirectional shadowing experiments show that under the same conditions the height of the chromatin fragments increases with the number of nucleosomes. These observations indicate that the electron microscope images studied in this work correspond to a top view of small chromatin fragments. Rotary-shadowed chromatin fragments show three parts: (a) a contour with a heavy deposition of platinum; (b) an annular zone between the central region and the periphery; and (c) a central hole. The heterogeneous ring generated by the deposition of platinum in the periphery suggests that nucleosomes form a one-start helix (5-7 nucleosomes per turn) that apparently can be left- or right-handed. The annular region (thickness of about 11 nm) shows spokes probably due to flat faces and core DNA of radially oriented nucleosomes. The central hole (8-12 nm) is clearly seen in many images but it is not empty because some deformed fragments show coated material (probably linker DNA) that protrudes from this central depression. We have observed that these structural elements directly detected in short chromatin fragments are also present in long chromatin fibers. This allows us to conclude that these elements are basic structural components of the 30 nm chromatin fiber.

scholarly journals Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

1983 ◽  
Vol 96 (4) ◽  
pp. 1132-1137 ◽  
Author(s):  
J R Paulson ◽  
J P Langmore
Keyword(s):  
Diffraction Pattern ◽  
Isolation Procedure ◽  
X Ray Diffraction ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
X Ray ◽  
Low Concentrations ◽  
Chromosome Isolation ◽  
Chromatin Fibers ◽  
High Concentrations

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.

Direct evidence for opposite effects of D-glucose and D-glyceraldehyde on cytoplasmic pH of mouse pancreatic β-cells

1986 ◽  
Vol 6 (4) ◽  
pp. 355-361 ◽  
Author(s):  
Per Arkhammar ◽  
Per-Olof Berggren ◽  
Patrik Rorsman
Keyword(s):  
Direct Evidence ◽  
Cytoplasmic Ph ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
High Concentrations

The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH4+ and high concentrations of K+ on cytoplasmic pH (pHi) were investigated in dispersed and cultured pancreatic β-cells from ob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pHi value in resting β-cells was 6.71. Addition of 20 m M of the physiological stimulus D-glucose increased pHi with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pHi. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pHi. In contrast to D-glucose, 10m M of D-glyceraldehyde decreased pHi by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pHi in pancreatic β-cells.

scholarly journals Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels.

1988 ◽  
Vol 106 (3) ◽  
pp. 657-666 ◽  
Author(s):  
F Di Virgilio ◽  
B C Meyer ◽  
S Greenberg ◽  
S C Silverstein
Keyword(s):  
Direct Evidence ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Fura 2 ◽  
Monolayer Cultures ◽  
Low Concentrations ◽  
High Concentrations ◽  
Macrophage Populations ◽  
Mouse Peritoneal Macrophages

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.

Mixing of brine with oil triggered sphalerite deposition at Pine Point, Northwest Territories, Canada

Geology ◽  
2020 ◽  
Author(s):  
Marko Szmihelsky ◽  
Matthew Steele-MacInnis ◽  
Wyatt M. Bain ◽  
Hendrik Falck ◽  
Robin Adair ◽  
...  
Keyword(s):  
Northwest Territories ◽  
Direct Evidence ◽  
Chemical Interaction ◽  
Fluid Mixing ◽  
Sulfide Mineralization ◽  
Principal Role ◽  
Pine Point ◽  
High Concentrations

Hydrocarbons are commonly invoked as triggers for the precipitation of sphalerite in carbonate-hosted Pb-Zn deposits, but direct evidence for the presence of petroleum during sulfide mineralization is rarely documented. Here, we report evidence of fluid mixing between basinal brines and oil during deposition of coarse sphalerite at a classic carbonate-hosted Pb-Zn district, Pine Point, Northwest Territories, Canada. The brines contain high concentrations of Pb, detectable aqueous sulfate, and hydrocarbons that attest to chemical interaction with oil. The oil inclusions in sphalerite contain much less Pb relative to the brines and no evident H2S, suggesting that the principal role of hydrocarbons was as a reductant. Mixing of brine with oil enabled the conversion of aqueous sulfate to sulfide, and thereby triggered sphalerite deposition.

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