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The effect of histone acetylation on the topology of plasmids transfected into COS7 cells was examined. Parallel determinations of histone profiles and DNA topology showed that with increasing levels of acetylation the minichromosomal DNA is gradually relaxed. This effect could not be attributed to the increased transcriptional activity accompanying butyrate treatment since plasmids with different promoter strengths exhibited similar superhelical densities. Considering that the number of nucleosomes/minichromosome were constant under these conditions, the data suggest that in vivo histone hyperacetylation reduces the linking number change/nucleosome.

The extrachromosomal DNA coding for the ribosomal precursor in Tetrahymena contains a transcribed region with a size of 6 x 10(3) base pairs plus non-transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P-labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r-chromatin are hypersensitive to DNase I. The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3' end. The hypersensitivity of the central part of r-chromatin is also found by autodigestion in isolated nucleoli.

Replicative intermediates of discrete size (approximately 41 S) are observed in the eukaryotic organism Tetrahymena pyriformis, when the organism is grown under defined physiological conditions. The intermediates (believed to represent replicons) are synthesized and accumulated over longer periods of time (less than 90 min), if the cells are treated with low concentrations of cycloheximide. Under these conditions the rate of total DNA synthesis is only slightly inhibited (less than 15%), while maturation of intermediate DNA into high-molecular-weight DNA is completely blocked (greater than 98%). Cycloheximide appears to inhibit the maturation process more specifically than other protein synthesis inhibitors. Studies of the accumulated intermediates on alkaline buoyant density gradients demonstrate that initiation of new putative replicons occurs during treatment with cycloheximide.

Inhibition of DNA synthesis in the eukaryotic organism Tetrahymena pyriformis by thymine starvation results in the formation and accumulation of a distinct class of DNA during the first hour after re-initiation of the synthesis. This DNA, when synthesized in the presence of 5-bromodeoxyuridine (BrdUrd), bands as a distinct peak at an intermediate density of (see article) in a neutral CsCl buoyant density gradient. Following short pulses of BrdUrd, the DNA of intermediate density accounts for up to 90% of the newly synthesized DNA and binding assays on nitrocellulose filters show the presence of single-stranded regions. Alkaline buoyant density and alkaline velocity gradients clearly demonstrate the presence of non-covalently linked newly synthesized fragments with an average length shorter than that of the parental strands. Pulse experiments show that the DNA of intermediate density is progressively converted to fully hybrid (light - heavy) DNA molecules. It is therefore suggested that in response to damage caused by thymine starvation, a replicative DNA intermediate accumulates at the growing point. A similar phenomenon has also been observed after irridation with ultraviolet light. The effect of the protein synthesis inhibitor cycloheximide on the formation on the intermediate and the fully hybrid material is also described.