Actinomycin binding of normal and phytohaemagglutinin stimulated lymphocytes (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.
Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes
Experimental Cell Research, 1973
The DNA-dependent RNA polymerase activity of isolated nuclei from human peripheral blood has been shown to increase following stimulation with phytohaemagglutinin (PHA). Using the toxin cr-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a twofold increase in the amanitin-resistant polymerase activity (polymerase A) with little increase in the sensitive polymerase activity (polymerase B). 24 h following PHA stimulation the amanitin-resistant activity is stimulated 4-5 fold and the amanitin-sensitive activity less than twofold. The susceptibility of this increased amanitin-resistant activity to low doses of actinomycin D both in vivo and in vitro indicates that the amanitin-resistant enzyme is mainly engaged in ribosomal RNA precursor synthesis. These changes in DNA-dependent RNA polymerase activity closely correspond to the observed changes in ribosomal and non-ribosomal RNA synthesis following lymphocyte stimulation. The increased polymer&e A activity is diminished by a 1 h incubation of the cells with cycloheximide added 24 h after PHA whereas polymerase B activity remains unaffected. This indicates that the polymerase A activity observed after transformation-is dependent on continuing protein synthesis. In our incubation conditions the polymerase activity observed in isolated nuclei appeared to be almost wholly attributable to elongation of nascent RNA molecules attached to the endogenous DNA template.
Experimental Cell Research, 1975
It has previously been shown that actinomycin D at a concentration of 0.08 pg/ml preferentially inhibits nucleolar (ribosomal) RNA synthesis in monolayer cultures of Chinese. hamster cells. Treatment with 0.08 rg/ml actinomycin D of Chinese hamster cells, synchronized by mitotic selection, at the beginning of the G 1 period causes a delay in the onset of DNA synthesis while a similar treatment in the late Gl period does not affect the process. The same effect has been produced by 9 pg/ml lucanthone (miracil D), a drug selectively inhibiting ribosomal RNA synthesis. The addition of 0.08 pg/ml actinomycin D to the stationary cultures stimulated to proliferate by medium changes completely prevents cells from entering the S period, when the drug is added to the medium in the first half of the pre-replicative period. However, in the second half of the prereplicative period cells regain the ability to synthesize DNA in the presence of actinomycin D approximately to the same extent as do cells in the G 1 period directly following mitosis. Thus, the demand for a de novo ribosomal RNA (rRNA) synthesis may be considered as a specific feature of cells induced to proliferate after a period of quiescence. The data obtained are consistent with the assumption that the first part of the pre-replicative period in stimulated quiescent cells is characteristic of GO cells while later on the GO cells enter the path of G 1 cells typical for the rapidly proliferating cell populations.
1969
For the purpose of revealing whether AMD inhibits the RNA synthesis of erythroblasts in an effective dose in vivo to eradicate erythroid cells in rabbit bone marrow, the author observed the RNA synthesis by H3-uridine incorporation in vitro and RNA level on the cells from the anemic animals taken at a certain period after a single injection of AMD in a small dose of 50 and 100μg/kg body weight. The data revealed that by such a small dose of injection of AMD the RNA synthesis of erythroid precursors, early basophilic and proerythroblast stages, was successfully suppressed without any suppressing effect on the RNA synthesis of erythroblasts in the later stages of specialization, indicating that there are at least two kinds of RNA synthesis: one seen mainly in the earlier stages of specialization and the other one seen mainly in the later stages, and they can be distinguished from each other by the AMD sensitivity.</p
Studies on the nature of messenger RNA in phytohemagglutinin-stimulated rat lymphocytes
Biochimica et biophysica acta, 1971
Ribonucleic acid synthesis during phytohemagglutinin-induced transformation of rat lymphocytes was investigated by deoxyribonucleic acid • ribonucleic acid hybridization and hybridization competition. It was found that the amount of ribonucleic acid complementary to deoxyribonucleic acid varied as a function of time after stimulation of lymphocytes with phytohemagglutinin. Ribonucleic acid synthesized between 19 and 24 h in stimulated lymphocytes showed the lowest hybridizability (approx. 2-3 °/o of the deoxyribonucleic acid), whereas ribonucleic acid synthesized either before or after this period showed a greater extent of hybridization. Hybridization-competition experiments demonstrated that unlabeled small-lymphocyte ribonucleic acid was a successful competitor in the reaction between radioactive transformed-lymphocyte ribonucleic acid and lymphocyte deoxyribonucleic acid. This suggests that resting small lymphocytes and transformed lymphocytes possess similar species of ribonucleic acid.
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.
Changes in extranucleolar transcription during actinomycin D-induced apoptosis
Histology and histopathology, 2005
Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 microg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromo-uridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar...
European Journal of Biochemistry, 1978
Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4-6 h following application