Biomedical subjects
R Klingholz
Publications and source records attributed to R Klingholz.
Differences among chicken erythrocyte histones H1 and H5 in associating with H1-depleted polynucleosomes.
1. Chicken erythrocyte histones H1a, H1b and H5 were associated to H1-depleted polynucleosomes from rat liver and the products were probed by digestion with micrococcal nuclease. 2. The digestion response of complexes with purified H1a or H1b resembled that of native polynucleosomes, while the digestion response of complexes with purified H5 exhibited specific different features--but none of these complexes resembled those with unfractionated histones H1a, H1b and H5. 3. However, after mixing purified histones H1a, H1b and H5 in the proportions (0.15:0.25:0.9) as these occur in erythrocyte nuclei and associating this mixture, the digestion response of the complexes was similar to that of the complexes with unfractionated histones.
Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes.
We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.
Reassociation of histone H1 to H1-depleted polynucleosomes.
A new procedure is described for the reassociation of histone H1 to rat liver polynucleosomes selectively depleted of H1 and nonhistone proteins. The fidelity of reconstitution was scrutinized by nuclease digestion and sedimentation studies monitoring the regeneration of structural features which disappear following removal of H1. The results demonstrate that the 166-base pair barrier of digestion with micrococcal nuclease was restored in polynucleosomes reconstituted at the same ratio of H1 per octamer as that found in native nuclei, while association of twice that amount of H1 produced a barrier of digestion at about 178-180 base pairs. Polynucleosomes associated with polylysine exhibited a similar barrier as H1-depleted polynucleosomes. Digestion of H1-reconstituted polynucleosomes with DNase I produced preferentially dinucleosomal DNA fragments in the same manner as that of untreated polynucleosomes. The rate of digestion of H1-reconstituted polynucleosomes by micrococcal nuclease and DNase I was also comparable to that of untreated polynucleosomes. Sedimentation of H1-reconstituted polynucleosomes in a quasi-physiological ionic milieu revealed the regeneration of disassembly-refractory particles, a structural feature of untreated polynucleosomes. We conclude that the nucleosomal and supra-nucleosomal structure of polynucleosomes was reconstituted with fidelity.
Digestion of chromatin to H1-depleted 166 basepair particles by Ca2+/Mg2+-dependent endonuclease.
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Supranucleosomal structure of chromatin: digestion by calcium/magnesium endonuclease proceeds via a discrete size class of particles with elevated stability.
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Structure of rat liver nuclei after depletion and reassociation of histone H1.
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Technology and production of man-made mineral fibres.
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