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T Koller

Publications and source records attributed to T Koller.

At least 55 records · Page 3Linked to original sources
PubMed1986

The effect of thyrotropin and cAMP on DNA synthesis and cell growth of human thyrocytes in monolayer culture.

Monolayer cultures of human thyrocytes from normal tissue (n = 10), and adenomas (n = 7), differentiated (n = 4), poorly differentiated (n = 2), and undifferentiated (n = 3) thyroid cancers were established to assess the significance of thyrotropin (TSH) and cAMP (adenosine 3',5'-cyclic monophosphate) on cell growth and DNA (deoxyribonucleic acid) synthesis. Cell growth of thyrocytes from normal and adenomatous tissues increased more rapidly (p less than 0.01) after TSH (0.1 IU/ml) was added but was unaffected by cAMP (10(-4) mol/L). In these cells, TSH also enhanced DNA synthesis twofold to twelvefold (p less than 0.01). The adenylate cyclase (AC) inhibitor, 2',3' dideoxyadenosine (ddA), increased DNA synthesis 1.3 to 6 times at a concentration of 2 X 10(-4) mol, whereas the membrane/passable cAMP analogue, dibutyryl-cAMP, and the AC stimulator, forskolin, failed to show any effect on DNA synthesis up to a concentration of 10(-5) mol/L (p less than NS). When administered simultaneously, TSH (1/2 maximum) and ddA (20 mumol) had no cumulative effect on DNA synthesis (p = NS). TSH stimulation in cancerous thyroid tissue (n = 11) demonstrated a lack of TSH response in seven of 11 monolayer cultures with no apparent correlation to cancer differentiation, patient age, or sex. Thus TSH was demonstrated to stimulate DNA synthesis and cell growth of human thyrocytes in monolayer cultures independent of the AC system. However, the TSH effect on cell growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues.

Adenoma↗
PubMed1985

Duplex DNA knots produced by Escherichia coli topoisomerase I. Structure and requirements for formation.

We investigated systematically the knotting of nicked circular duplex DNA by Escherichia coli topoisomerase I. Agarose gel electrophoresis of knots forms a ladder of DNA bands. Each rung is made up of a variety of knots with the same number of nodes, or segment crossings; knots in adjacent rungs differ by one node. We extended the technique of electron microscopy of recA protein-coated DNA to the visualization of the complex knots tied by topoisomerase I. The striking result is that the enzyme produces every knot theoretically possible. The requirement for excess enzyme to form complex knots suggests a role for topoisomerase I in contorting the DNA in addition to promoting strand passage. We conclude that nodes formed are equally likely to be positive or negative and that topoisomerase I can pass DNA strands through a transient enzyme-generated break without regard to orientation of the passing strand. The results are interpreted in terms of a formulation for the topological requirements for knotting.

Bacteriophage phi X 174↗
PubMed1984

Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin.

Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells. The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy. Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions. The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking. The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen. Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA. This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen. Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region. In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer. In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy. There was no difference in the appearance or the frequency of the transcription units seen. Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA. Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin. We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Nucleolus↗
PubMed1984

Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin.

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.

Animals↗
PubMed1984

Involvement of the globular domain of histone H1 in the higher order structures of chromatin.

We have attacked H1-containing soluble chromatin by alpha-chymotrypsin under conditions where chromatin adopts different structures. Soluble rat liver chromatin fragments depleted of non-histone components were digested with alpha-chymotrypsin in NaCl concentrations between 0 mM and 500 mM, at pH 7, or at pH 10, or at pH 7 in the presence of 4 M-urea. alpha-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by alpha-chymotrypsin can be easily monitored. The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: under conditions of H1 dissociation at 400 mM and 500 mM-NaCl (pH 7 and 10); at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; in the presence of 4 M-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mM and 300 mM-NaCl. Under these conditions H1 is degraded by alpha-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin. Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for alpha-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

Animals↗
PubMed1984

A structural concept for nucleoli of Dictyostelium discoideum deduced from dissociation studies.

We aimed to establish whether there is a matrix structure in the nucleolus to which the ribosomal DNA (rDNA) is strongly attached. To detect artifacts that might occur during the harsh histone extraction procedures frequently used for matrix preparation, we dissociated nucleoli of Dictyostelium discoideum with a range of NaCl or heparin concentrations. With heparin treatment significant amounts of rDNA were solubilized into the dissociating solution. When the residual nucleoli were digested with Eco RI, none of the Eco RI fragments of the rDNA remained preferentially bound to the residual nucleoli, indicating that there is no matrix attached to a specific site on the rDNA. When residual nucleoli were examined by electron microscopy, a correlation was found between the extent of solubilization of rDNA, the loss of nucleosomes, and, in heparin-treated nucleoli, the loss of ribonucleoprotein-bound components. These results suggest that the rDNA is released from the nucleoli as soon as nucleosomes have been dissociated and transcription complexes disrupted. Electron microscopy also showed that the NaCl concentration required for dissociation of nucleosomes was higher when divalent cations (Ca2+, Mg2+, Cu2+) were used during the isolation or the treatment of the nucleoli prior to dissociation in high salt. Furthermore, the residual, high-salt-resistant structures were much larger when nucleoli were pretreated with divalent cations or when they were purified in the presence of Ca2+ than when they were purified in its absence. Hence divalent cations, which induce chromatin condensation, prevented nucleolar dissociation whereas treatment with chelating agents, which loosen chromatin compaction, led to much smaller residual matrixlike structures. Nucleoli could be dissociated with heparin to a larger extent than with NaCl so that in Ca2+-free preparations no residual nucleolar matrixlike structures could be detected. Our results suggest that the nucleolar "matrix" seen in the electron microscope is due to incomplete dissociation of the nucleolar material. We propose that in nucleoli of Dictyostelium the rDNA is not attached to a tightly binding matrix structure, but that nucleoli are stabilized by side-to-side contacts between chromatin fibers and transcription complexes.

Cell Fractionation↗
PubMed1983

Involvement of the domains of histones H1 and H5 in the structural organization of soluble chromatin.

We have studied in reconstitution experiments the conditions under which peptides derived from histones H1 and H5 are bound in chromatin and to what extent they are involved in the organization of chromatin fibers. The fragments of rat liver histone H1 (rH1) and chicken erythrocytes H1 (cH1) and H5 (cH5) used were the globular domains (rG-H1, cG-H1, cG-H5), the globular domain and the N-terminal tail (rCT-N), about half of the globular domain and the C-terminal tail (rNBS-C) and the C-terminal tail (rCT-C). Fragments containing the C-terminal tail (rNBS-C and rCT-C) dissociate from H1-depleted rat liver chromatin at 300 mM-NaCl and above (similar to uncleaved H1) and fragments lacking the C-terminal tail (rG-H1 and rCT-N) dissociate between 100 and 200 mM-NaCl. This suggests that at putative physiological ionic strengths the binding of rH1 is dominated by its C-terminal tail, whereas the globular region and the N-terminal tail might only be loosely bound or not bound at all and by this modulate chromatin structure. The globular domain of cH5 binds more tightly than that of the chicken and rat H1 and is only partially released at 200 mM. Since in the transcriptionally silent erythrocytes of birds H5 replaces H1 to a large extent, we suggest that the globular domain of H1 serves as a temporary seal and that of H5 as a permanent seal of the nucleosome. All the H1 and H5 peptides tested condensed and precipitated chromatin and H1-depleted chromatin: rNBS-C and rCT-C at lower peptide per nucleosome ratios than rG-H1, cG-H1 and rCT-N. At about one peptide per nucleosome none of the H1 fragments induced condensation similar to that of native chromatin. At a peptide per nucleosome ratio close to the point of precipitation, all H1 fragments, but not poly-L-lysine, induced similar compact forms which were fiberlike, although more irregular than the compact fibers of native chromatin. These reconstitution experiments suggest that both halves of H1 as well as the globular domain by itself are involved and capable in forming higher-order chromatin structures. Details of these structures are not known.

Animals↗
PubMed1983

Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation.

The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.

Cell Nucleolus↗
PubMed1983

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding.

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z-like contribution and antibodies raised against Z-DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine-pyrimidine sequences and GC base pairs.

Animals↗
PubMed1982

Determination of the number of superhelical turns by the hyperchromicity of partially denatured covalently-closed DNA molecules.

A novel method of determining the number of superhelical turns of a covalently-closed plasmid DNA is described. It relies on the determination of the hyperchromicity, and hence the proportion of unstacked basepairs, of a partially heat-denatured sample which co-migrates during electrophoresis with nicked circular duplex DNA. The values obtained for plasmid pBR beta G DNA at 4 degrees C (-29.8 and -33.5 in the two buffers used) agree closely with the values obtained in parallel by topoisomer band-counting. Our method is less precise than band-counting but is readily applicable to determining the superhelicity of very large DNA molecules. Our results confirm earlier findings that magnesium-containing buffers cause an increase in the duplex winding angle, and hence an increase in the number of negative superhelical turns.

Animals↗
PubMed1982

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus.

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 micrometers. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).

Animals↗