Actinomycin D-binding in vivo: active chromatin preferred

Escherichia coli RNA polymerase and the endogenous engaged RNA polymerase I were used as specific probes to monitor the physiologically inactive and active nucleolar chromatin template function, respectively. Actinomycin D bound preferentially to the physiologically active regions of rat liver nucleolar chromatin in vivo.     

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

Actinomycin D peritonitis in the rat.

Following intraperitoneal injection of actinomycin D rats show a decrease in number of cells present in the peritoneal cavity, reaching the lowest point after 24 hr. At the same time a highly significant increase of free beta-glucoronidase and of the intracellular concentrations of both cyclic AMP and cyclic GMP has been observed. No exudate was present at this time. Measurable quantities of exudate were present 48-72 hr after actinomycin injection concomitantly with an intense cellular immigration, the dominant cell being mononuclears. In this second phase of the reaction the free beta-glucuronidase decreases towards normal values and both the cyclic nucleotides are significantly below the control values. It is suggested that the increase of intracellular cAMP--concomitant with the maximum release of lysosomal enzymes--is a feedback mechanism preventing further release of inflammatory mediators.     

Actinomycin D binding to DNA and chromatin: A colorimetric procedure suitable for the analysis of turbid preparations and for simultaneous processing of several samples

A simple, sensitive, and inexpensive method is presented for measuring the binding of actinomycin D (AMD) to either chromatin or DNA. The procedure gives results which are comparable with those obtained by the optical titration procedure, but is superior to that method in that it does not require continuous attention, can be used with turbid chromatin preparations, and allows simultaneous processing of several samples. Chromatin or DNA that has been allowed to react with AMD is subjected to sucrose density gradient centrifugation to remove unbound antibiotic. The AMD-DNA or AMD-chromatin complex is then dissociated by incubation with 5m urea, and the released AMD is extracted with ether and estimated spectrophotometrically.     

Actinomycin D: Effects on Mouse L-Cells

The lethal and inhibitory effects of actinomycin D (Act D) on asynchronous and synchronized populations of mouse L-cells have been studied. It has been shown that the survival curve of populations in the logarithmic phase of growth can be approximated by two exponential survival curves corresponding to a sensitive and resistant moiety. The size and sensitivity of both moieties vary during the growth of the population. As the cell population moves through logarithmic and into stationary phase, the sensitive moiety becomes smaller but more resistant whereas the resistant moiety increases in size and also becomes more resistant. This variation appears to be related to a reduced uptake of Act D and also a reduced rate of DNA and RNA synthesis. Variations in sensitivity to the drug have also been observed during the division cycle of synchronized cells with cells in the S phase showing the greatest uptake of the drug and also the greatest sensitivity. However, no direct correlation between uptake and sensitivity has been established. Actinomycin D has inhibitory effects on both RNA and DNA synthesis. RNA synthesis is inhibited rapidly but does not seem to drop to less than 5% of the control value. The inhibition of DNA synthesis appears to occur over a longer period and may reach values as low as 0.25% of control. In both cases the degree of inhibitions appears to be dependent on both the length of exposure and the concentration of the drug. Certain similarities between the response of cells to Act D and X-rays have been observed and are discussed.     

放线菌素D诱导植物离体细胞染色体变异

用放线菌素D处理辣椒愈伤组织,明显抑制愈伤组织细胞的离体增殖,并诱发染色体数量与结构变异。细胞核DNA荧光染色测定,表明放线菌素D干扰了DNA合成复制,是导致离体细胞染色体变异的直接原因。     

Nuclease sensitivity of active chromatin.

The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression.     

Assembly of transcriptionally active chromatin in vitro: a possible role for topoisomerase II

Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.     

Production of Actinomycin D in Complex Media

The production of actinomycin D was examined using Streptomyces antibioticus growing on a complex medium based on glucose and casein hydrolyzate. Purification of the medium to remove calcium increased actinomycin production by about one-third and prevented production of a characteristic brown pigment. The sensitivity to magnesium concentrations was less than was reported by Katz et al.,¹¹⁾ while similar requirements were found for zinc and iron.     

Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors

Active minichromosomes assembled on injected 5S RNA gene clones are stable in Xenopus oocytes; endogenous 5S DNA specific factor(s) are required for their assembly. When somatic-type and oocyte-type 5S RNA gene clones are coinjected, the somatic genes are assembled into active minichromosomes, while most of the oocyte genes are assembled into inactive ones. The differential 5S RNA gene expression, which mimics that in somatic cells, appears to result from titration of 5S DNA specific factor(s) by the competing somatic 5S DNA, followed by histone mediated assembly of inactive chromatin on the oocyte 5S DNA. Stable minichromosomes are also assembled on a cloned histone H4 gene; again, intragenic DNA rearrangements affect the efficiency of assembly of active chromatin and differential gene expression occurs after coinjection of two or more H4 DNA constructs. We suggest that the H4 DNA molecules also compete for limiting quantities of specific DNA binding factor(s) required for the assembly of active H4 gene chromatin.