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T Koller

Publications and source records attributed to T Koller.

At least 73 records · Page 4Linked to original sources
PubMed1982

Involvement of higher order chromatin structures in metaphase chromosome organization.

Using electron microscopy we show that the metaphase chromatin fibers of Chinese hamster ovary cells form the same ionic strength-dependent higher order structures as the corresponding interphase chromatin fibers. We present evidence that such intact chromatin fibers are a prerequisite for the maintenance of the characteristic shape of metaphase chromosomes. The evidence is based on the finding that treatment of chromosomes with 0.5 M NaCl, a condition which is known to remove histone H1 and which destroys the higher order structure of chromatin fibers, also leads to a disintegration of the metaphase chromosome structure, whereas treatment with 0.3 M (or less) NaCl has no effect on the integrity of the chromosomes and their chromatin fibers. These data support a model in which the metaphase chromosome is maintained by a tight assembly of the 25-30 nm thick chromatin fibers containing all the histones.

Animals↗
PubMed1982

Structure of the active nucleolar chromatin of Xenopus laevis Oocytes.

Active nucleolar chromatin of Xenopus laevis oocytes was prepared for electron microscopy by a step gradient method, which separates the chromatin from proteins and other constituents that might nonspecifically bind at low ionic strength. Between putative RNA polymerases and within the nontranscribed spacer region, the chromatin appears as smooth, thin filaments. For the first time, it is shown here that these filaments are indistinguishable from pure DNA absorbed to the same specimen, even when the ionic strength is raised up to 100 mM NaCl. Bulk rat liver chromatin, however, which was coprepared as a biochemically well-characterized standard with the active nucleolar chromatin, shows nucleosomes containing fibers, which condense into supranucleosomal structures with increasing ionic strength. Since the appearance and the behavior of active nucleolar chromatin at different ionic strengths and pHs resembles tht of pure DNA, but not of any known type of chromatin, it is suggested that, except for the transcription apparatus, very few macromolecular constituents are associated with ribosomal DNA during transcription. The observations described in this paper explain most of the published and partly conflicting results obtained by electron microscopy of nucleolar chromatin.

Animals↗
PubMed1982

Structure of the active nucleolar chromatin of Xenopus laevis oocytes.

Active nucleolar chromatin of Xenopus laevis oocytes was prepared for electron microscopy by a step gradient method, which separates the chromatin from proteins and other constituents which might unspecifically bind at low ionic strength. Between putative RNA polymerases and within the non-transcribed spacer region, the chromatin appears as smooth, thin filaments, indistinguishable from pure DNA adsorbed to the same specimen. These filaments are found under all conditions tested, even in the presence of 100 mM NaCl. On the other hand, bulk rat liver chromatin, which was co-prepared with the active nucleolar chromatin, shows nucleosomes containing fibers, which condense into supranucleosomal structures with increasing ionic strength. Since the appearance and the behaviour of active nucleolar chromatin at different ionic strength and pH resembles that of pure DNA, but not that of any known type of chromatin, it is suggested that, except for the transcription apparatus, few macromolecular constituents are associated with ribosomal DNA during transcription.

Animals↗
PubMed1981

Electron microscope specimen preparation of rat liver chromatin by a modified Miller spreading technique.

We describe a modification of the spreading method of Miller and Bakken [17]. Prior to mounting the samples, the carbon supports used for electron microscopy are coated with Alcian blue. By this method the chromatin fibres stick firmly to the supporting film such that the shape of the fibres is independent of the washing and drying procedures used during specimen preparation. Using this technique we show an ionic strength-dependent condensation of rat liver chromatin protruding from lysed nuclei which is fully compatible with that reported previously [34] for soluble chromatin.

Adhesiveness↗
PubMed1981

Structural changes of soluble rat liver chromatin induced by the shift in pH from 7 to 9.

Soluble rat liver chromatin was studied at pH 7 and at pH 9. In order to remove selectively non-histone components or non-histone components and histone H1, fractionation of chromatin was performed at pH 7 and pH 9 at different ionic strengths. The salt-dependent condensation of the fractionated chromatin was analysed in the electron microscope. There is no difference between the appearance of H1-depleted chromatin at poH 7 and pH 9. In H1-containing chromatin the shift from pH 7 to pH 9 leads to the following morphological changes: a) at very low ionic strength the nucleosomes unravel partially or totally and the zigzag-shaped fibres disappear in favour of beads-on-a-string; b) with increasing ionic strength the filaments condense into fibres, however, these fibres appear distorted and clearly less ordered than at pH 7. There is no indication of a release or displacement of histone H1. The pH-effect is completely reversible. The data suggest a pH-induced change in the mode of action of histone H1 in the formation of nucleosome beads and higher order chromatin structures.

Animals↗
PubMed1979

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.

Bacillus subtilis↗
PubMed1979

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.

Chromatin↗
PubMed1978

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.

Coliphages↗
PubMed1978

Electron microscopic determination of the binding sites of ribosomal proteins S4 and S8 on 16S RNA.

Specific complexes.early in the assembly of Escherichia coli ribosomes were examined in the electron microscope. Complexes between ribosomal protein S4 or S8 and 16S RNA were fixed gently with formaldehyde and then denatured for protein-free spreading. Binding of each protein was found to preserve an easily recognized configuration in the RNA that allows the sites of protein binding to be determined. S8--16S RNA complexes have a single hairpin loop near the middle of the 16S RNA, 798 +/- 21 bases from one end and 657 +/- 26 bases from the other. S4-16S RNA complexes have two adjacent loops at one end with 250--450 bases. This structure probably arises from the simultaneous binding of S4 to three noncontiguous sites on the RNA. Measurements of these complexes place the binding sites near the 5' end, at more than one site 250--585 nucleotides from the 5' end and 645 +/- 45 bases from the 3' end. The latter site has not been recognized previously as a distinct S4 binding site. This approach allows the binding sites to be determined without knowledge of the nucleotide sequence and gives insight into the configuration of the rRNA in the assembling ribisome.

Binding Sites↗
PubMed1977

Influence of histone H1 on chromatin structure.

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.

Animals↗
PubMed1976

Superstructure of linear duplex DNA.

The superstructure of a covalently closed circular DNA (of bacteriophage PM 2) was compared by electron microscopy with that of a linear duplex DNA (of bacteriophage T7) when ionic strength and benzyldimethylalkylammonium chloride concentration were varied. In parallel studies the sedimentation behavior of these DNAs was studied by analytical ultracentrifugation, but for technical reasons these had to be without benzyldimethylalkylammonium chloride. By combining the information from the two methods one has to conclude that with increasing ionic strength the linear duplex T7 DNA spontaneously forms a structure similar to that of the superhelical structure of closed circular PM 2 DNA. The superstructure is destroyed under premelting conditions and in the presence of an excess of ethidium bromide.

Coliphages↗
PubMed1975

A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules.

A protein-free nucleic acid preparation method for electron microscopy is described. The basic procedure is very similar to the classical protein monolayer spreading techniques. The carrier protein (usually cytochrome c) is replaced by benzyldimethylalkylammonium chloride. Both the hypophase method and the microdiffusion or droplet method can be applied with this compound. Unlike cytochrome c, benzyldimethylalkylammonium chloride does not lead to any apparent thickening of the nucleic acid strands. Partially denatured DNA spread with this reagent shows a loosened structure with a foamy appearance in the regions previously considered to be "unmelted," which open up locally into melted loops of different size. Specifically bound proteins, such as RNA polymerase on bacteriophage T7 DNA, can be detected unambiguously.

Coliphages↗
PubMed1975

Adsorption of DNA molecules to different support films.

Protein-free adsorption of the DNA of the Escherichia coli bacteriophage T7 to carbon, collodion, aluminium-beryllium and aluminium films was studied. It was found that the appearance of DNA strands depended greatly upon the kind of support film used. Direct adsorption of DNA to aluminium-beryllium or aluminium films yielded specimens with 'thin and long' and 'thick and short' regions along the strand. Well extended, uncoiled and unaggregated DNA molecules were obtained only when DNA was adsorbed to carbon, collodion or mica in the presence of intercalating dyes such as ethidium bromide. Adsorption properties of the different films are well correlated with their surface charge. Aluminium-beryllium films carry a strong positive surface charge, aluminium films a weak positive charge and carbon films a weak negative charge. It is suggested that for the preparation of specimens by spontaneous adsorption of well extended and unaggregated strands it is necessary that the DNA molecule is stiffened by a ligand such as an intercalating dye, and that the charge on the surface of the support film is opposite to the charge of the macromolecule.

Adsorption↗