Effects of low concentrations of actinomycin D on the initiation of DNA synthesis in rapidly proliferating and stimulated cell cultures (original) (raw)
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The Journal of Cell Biology, 1975
The reinitiation of the synthesis of major RNA species has been studied in 37 RC cells after maximal inhibition of RNA synthesis by actinomycin D (AMD). During the period of recovery from AMD, resynthesized RNA (rec-RNA) is initially composed of almost exclusively light (4-14S) heterogeneous RNA species. All normal species of RNA can be detected in the rec-RNA spectrum as early as 3 h after AMD removal. The synthesis of low molecular weight methylated RNA species increases slightly during the early period after AMD removal, while the increase of low molecular weight unmethylated species is more significant during the same period. Much of the radioactivity in the polyribosomal fraction is EDTA and puromycin sensitive. Since polysomal, puromycin-sensitive RNA is polyadenylated (as evidenced by the binding to poly-U filters), and is heterogenous in size, it belongs to the m-RNA class. The synthesis of m-RNA increases immediately after AMD removal, whereas the reinitiation of the r-RNA synthesis occurs after a lag period of about 2 h. The kinetics of recovery of the synthesis of major RNA species from AMD inhibition show a size dependency comparable to the size-related sensitivity to AMD inhibition in other cellular systems. This dependency is most clearly seen in HnRNA, the AMD sensitivity of which is measured by the length of the lag period between AMD removal and the appearance of HnRNA fractions in a sucrose density gradient. Low molecular weight HnRNA reappears first, whereas heavier fractions of HnRNA appear in the spectrum after a lag period, the length of which is in direct relation to the position of the HnRNA fraction in the gradient.
Proceedings of The National Academy of Sciences, 1962
Actinomycin D is one of a number of polypeptide antibiotics isolated in Waksman's laboratory.1' 2 Bacteriostatic effects, particularly on gram positive bacteria, and antitumor activity have been attributed to this compound.2 3 Kirk4 has demonstrated that the addition of actinomycin D (0.2 to 0.5 ,4M) to exponentially growing cultures of Staphylococcus aureus stops RNA synthesis immediately. This effect is rapidly followed by an inhibition of protein synthesis, and later by a partial inhibition of DNA synthesis. The action of this compound is not related directly to energy production since both respiration and glycolysis of inhibited cells are unaffected by concentrations up to 0.1 mM.4 Kirk also demonstrated that the combination of DNA and actinomycin D results in a spectral change of the latter compound. These observations suggest the formation of a complex between these two compounds since Kawamata and Imanishi5 found no interaction of actinomycin and RNA and the reaction appears to be relatively specific for DNA. Although Rauen et al.6 have reported complex formation between actinomycin and RNA, 100 times more RNA than DNA is required.
TIMING OF DNA SYNTHESIS IN THE MITOTIC CYCLE IN VITRO
The Journal of Biophysical and Biochemical …, 1961
A study was made of the timing of DNA synthesis in the mitotic cycle under conditions where the average mitotic cycle of populations of human amnion and kitten lung cells in culture was variable. Three types of experiments were performed: (a) Autoradiographs were made of incorporated tritiated thymidine in cells whose mitotic histories were recorded microcinematographically allowing the measurement of telophase + G1 along with the total length of the mitotic cycle. (b) Measurement of the G2 + prophase part of the mitotic cycle was performed under various conditions by exposing cells to tritiated thymidine and observing the increase in labeled metaphases plus anaphases as a function of time. (c) The G2, the period from the end of DNA synthesis to prophase.
Relationship Between Rna Synthesis, Cell Division, and Morphology of Mammalian Cells
Journal of Cell Biology, 1966
Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P32 and H3-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division ...
Site of actinomycin-resistant RNA synthesis in animal cells
Experimental Cell Research, 1973
RNA synthesized in animal cells in the presence of high doses of actinomycin had many of the properties associated with hnRNA. The nucleoplasm was established as the site of this synthesis, as determined by radioautography and by differential extraction. No synthesis was observed in nucleoli. Therefore this RNA, which was unmethylated, did not represent abortively synthesized incomplete ribosomal RNA chains. Resistance to ethidium bromide also ruled out mitochondria as the site of synthesis of this RNA. RNA synthesized in the presence of actinomycin like hnRNA was resistant to inhibition by cordycepin. RNA synthesized in the presence of actinomycin contained a ribonuclease-resistant core. When first synthesized this RNA was associated with a high molecular weight RNA which was degraded rapidly. This unique species of RNA synthesized in the nucleoplasm of animal cells may exert a control function in the transcription of hnRNA in the cell nucleus or may play a role in the transport of mRNA from nucleus to cytoplasm.
Cell differentiation, 1985
Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.
Actinomycin D-resistant RNA synthesis in animal cells
Biochemical and Biophysical Research Communications, 1963
In attempts to determine the fate of rapidly-labelled nuclear RNA we have encountered a paradox which we believe is best explained by postulating that in animal cell nuclei there is, in addition to Actinomycin D-sensitive RNA synthesis (1) a system which is Actinomycin D-resistant and which may be a precursor of a rlbosomal component.
Regulation of ribosomal RNA synthesis in mammalian cells: effect of toyocamycin
Biochemistry, 1977
The present study shows that the antitumor agent toyocamycin (4-amino-5-cyano-7~-D-ribofuranosylpyrrolo(2-3d)pyrimidine) affects r R N A transcription in Ehrlich ascites cells. This action of the antibiotic is dependent on the amino acid composition of the cell culture medium. In cells incubated in a medium rich in amino acids, the high transcription rate of rRNA is lowered by the addition of 2 X M toyocamycin, while in amino acid starved cells the decreased level of rRNA synthesis remains unaffected. Processing of the
Studies on the Action of the Nuclear Factor Promoting Actinomycin D-Binding Capacity of Chromatin
Differentiation, 1977
It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.