Untersuchungen über die Regulation der DNS-Synthese während des Aktivitätswechsels vonAgrostemma githago-Samen

The relationships between DNA synthesis and germination capacity ofAgrostemma seeds have been studied. Protein synthesis and RNA synthesis are activated at the very beginning of imbibition, whereas DNA synthesis starts in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30° C), or aged seeds with a low germination capacity are characterized by a remarkably reduced protein synthesis. DNA synthesis is also reduced. The inhibition of protein-synthesis ofAgrostemma embryos fed with cycloheximid or actinomycin D causes a depression of DNA synthesis. These results indicate that the initiation of DNA synthesis of imbibingAgrostemma seeds depends on the synthesis of special proteins. Abscisic acid inhibits growth as well as DNA synthesis of isolatedAgrostemma embryos. Mitomycin inhibits germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also show a reduced incorporation of³H-thymidine in DNA. We suggest that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, is involved in the mechanism of afterripening of theAgrostemma seeds.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

ACTINOMYCIN D-INDUCED CHANGES IN GROWTH AND RIBONUCLEIC ACID METABOLISM IN THE GAMETOPHYTES OF BRACKEN FERN

Actinomycin D affected the morphological type of growth in the gametophytes of Pteridium aquilinum and the distribution of RNA and protein in their particulate fractions. Increasing concentrations of the drug progressively inhibited two-dimensional growth at the end of a period during which controls had formed typical two-dimensional plants. RNA was lost maximally from the nuclei-rich and ribosome-rich fractions of plants growing in a concentration of actinomycin D which inhibited two-dimensional growth. The magnitude of changes in protein content of the plants was less striking. Presence of actinomycin D in the medium also suppressed incorporation of uridine-H³ into cytoplasmic fractions of gametophytes. The possibility that two-dimensional growth in the gametophytes is under control of a newly synthesized messenger RNA, which is sensitive to actinomycin D, is discussed.     

CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G₂ (or G₂-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G₂ is 30-fold greater than it is in G₁. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.     

Inhibition by Actinomycin D of Uridine Diphosphoglucose Synthetase Activity During Differentiation of Dictyostelium discoideum

Actinomycin D inhibits the increase in specific activity of uridine diphosphoglucose synthetase during differentiation of the cellular slime mold, Dictyostelium discoideum. The degree to which this inhibition occurs is strictly correlated with, and may result from, the general inhibition of morphogenesis by actinomycin D.     

Resolution of electronic absorption bands and nucleotide binding processes in actinomycin D

Complexes of actinomycin D with model dexoxynucleoides have been studied by means of absorption spectroscopy and CD spectroscopy and CD spectroscopy. The CD spectra of the complexes of actinomycin D with 5′-dGMP, pdG-dT, pdT-dG, pdG-dA, and pdA-dG, respectively, all resemble one another. It is presumed that in solution the interactions between the guanine residues and actinomycin D in these complexes are the same as found in the crystalling 1:2 actinomycin D:dG complex [Jain, S. C. & Sobell, H. M. (1972) J. Mol. Biol. 68 , 1–20]. The CD spectrum of the Complexes with pdG-dC differs from of the complexes just mentioned, and resembles those of the complexes formed by actinomycin D with calf-thymus DNA and with poly(dG-dC)-poly(dG-dC). These resulls are consisent with, the nontion that pdG-dC froms a double-staranded intercalated complex with actinomycin D, and that the dG-dC sequence is an important binding site for actinomycin D in polydeoxynucleotides. Titrations of actinomycin D with monodeoxynucleotides were monitored at 380, 425, 440,465, and 480, nm in both absorption and CD modes. Comparisons fo saturation profiles at these wavelengths reveal that the curves obtained at various wavelenths do not superimpose with each other, so that they must reflect different titation processes. In complex formation wiht any given nucleotide, the apparent binding affinity monitored at these wavelengths decreases in the order given above. Based on these resulted and on features noted in the CD spectra of certain complexes, it is concluded that a minimum of theree electronic transitions underlie the visible absorption band of actinomycin D, extending earlier findigs. Comparing the titration proffiles obtained with dAMP and dIMP with the result for these systems in mmr titratins [Krugh, T. R. & Chen, Y. C. (1975) Biochemistry 14 , 4912–4922], it is concluded that one transition, centered at 370 nm, monitors preponderantly effects occurring at the 6 (benzenoid) nucleotide binding site and a second transition, located at 490 nm, is sensitive preferentially to processes occurring at the 4 ( quinoid) binding site. The latter is probably closely asscoiated with 2-amino and /or 3-carbonyl substituents. The third transition, identified with the absorption maxium at 420–440 nm, appears to reflect contributions arising in the entire phenoxazone chromophore. Using these band assignments, it is concluded that the benzenoid site binds nucleotides 1.5–3 times more avidly than does the quinoid site. CD titrations resolve these processes more effectively than do abscrption titrations. Aspects of the structures of these complexes formed in solution are discussed.     

The binding of actinomycin D and adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug:DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of Φχ174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.     

Control of in vivo protein synthesis and peroxidase formation by DNA-containing extracts of normal and crown gall tissues

Nucleic acids extracted from normal bean hypocotyl tissue (NE) and crown gall tumors (TE) affect amino acid incorporation into protein and the development of peroxidase activity when vacuum infiltrated into normal receptor tissues. TE enhances and NE inhibits both processes; NE from successively older tissues produces progressively greater inhibitions per unit of infiltrated nucleic acid. The active material has an absorption maximum at 257 nm with an A_260:280 ratio of more than 2·0. On acrylamide gel electrophoresis it shows a small DNA peak, four typical r-RNA peaks and a small low molecular weight RNA peak. Activity in such extracts is completely destroyed by hydrolysis with 0·3 N KOH or DNAase; RNAase is only slightly effective and pronase ineffective. It is deduced that the effective material contains DNA that may be complexed with RNA or other materials in the extract. Pretreatment of donor tissues with actinomycin d or 5-fluorouracil diminishes or annuls the activity of the extract. Pretreatment of receptor tissue with actinomycin d inhibits the action of TE but not of NE; pretreatment with cycloheximide prevents the action of both NE and TE.     

RNA synthesized during late sporulation is required for germ tube formation inBlastocladiella emersonii

Inhibition of RNA synthesis with actinomycin D as late as 210 min (T₂₁₀) afterBlastocladiella emersonii is induced to sporulate results in complete blockage of germ tube formation in the next generation. In agreement with other reports, actinomycin D added during germination did not block germ tube formation. Protein synthesis during germination is reduced by approximately one-half when actinomycin D is added atT₂₁₀ but remains at virtually control levels when actinomycin D is added during germination. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel analyses of the abundant proteins synthesizedin vivo during the first hour of germination revealed no qualitative differences in the proteins which accumulate when control cells are compared to cells treated with actinomycin D atT₂₁₀. Comparison of proteins synthesized from 20 to 40 min germination vs 40 to 60 min germination demonstrated that actinomycin D alters the temporal pattern of accumulation of some abundant proteins. The RNA synthesized afterT₂₁₀ is associated with polysomes, suggesting that an mRNA fraction made in late sporulation is required for a germ tube. The available data do not exclude the possibility that a regulatory RNA synthesized during late sporulation is required for germ tube formation.     

Interaction of 2-deamino- and 2-deamino-2-nitroactinomycin D with calf-thymus DNA and formation of ion-radicals

2-Deaminoactinomycin D (3a) and 2-deamino-2-nitroactinomycin D (2a) were prepared in one step from actinomycin D (1a, AMD) by reaction with nitrous acid. New DNA-binding (calf-thymus) data obtained by difference uv and CD spectra and ΔTm were presented. In vitro cell growth inhibitory activity of CCRF-CEM cells was also reported. The 2-deamino analog, 3a, does not bind to DNA strongly nor by intercalation of its chromophore. However, some binding with DNA was indicated by CD which is attributed only to hydrogen bondings of the peptides with the DNA helix; the affinity for binding is in the order 1a ? 2a > 3a. The 2-nitro analog, 2a, is a more potent agent against CCRF-CEM cells than the 2-deaminoactinomycin D, 3a; the potencies are in the order 1a > 2a ? 3a. Furthermore, the microsomes activate the analogs to free radical states which catalyze the production of superoxide, as indicated by electron paramagnetic resonance studies and oxygen consumption experiments.     

Invertase activity and cell growth in lentil epicotyls

The activity of invertase and its relation to growth were studied in the epicotyls of lentil seedlings incubated in the presence and absence of gibberellic acid (GA₃).

Invertase activity per epicotyl increases relatively more rapidly than does length, reaches a maximum during most active elongation, and declines upon cessation of growth.

GA₃ enhances both growth and increase in invertase activity, without altering the kinetics of the 2 processes. If GA₃ is added during incubation invertase activity increases more rapidly than does elongation rate.

Incubation of the seedlings in solutions of polyethyleneglycol inhibits the increase of both growth and invertase activity, the latter actually undergoing a decline, but causes no great change in the relative effect of GA₃. In presence of polyethyleneglycol GA₃ has however a relatively greater effect on invertase activity than on growth.

Sugars in the incubation medium have no significant effect on growth and invertase activity in the epicotyl, except inhibition at relatively high concentrations.

Cycloheximide, actinomycin D, and 5-fluorodeoxyuridine (FUDR) inhibit both growth and the increase in invertase activity. Added during incubation cycloheximide causes complete inhibition of growth and a decrease in invertase activity with no appreciable lag phase. With actinomycin D and FUDR the inhibition occurs after lag periods of 2 to 3 and of at least 10 hours, respectively. Thus the increase in enzyme activity is very probably based on de-novo synthesis, and the enzyme is in a state of turnover during growth.

The enzyme is present in soluble form in the cytoplasm, not firmly bound to any cell structures.

     

Phytochrome destruction: an apparent requirement for protein synthesis in the induction of the destruction mechanism

Examination of the phytochrome destruction reaction as a function of age in etiolated oat (Avena sativa L. cv. Garry) seedlings demonstrates that following illumination of 3-day-old shoots there is a lag, not observed in 4- or 5-day-old oats, prior to the onset of destruction. This light-mediated induction of the phytochrome destruction mechanism in 3-day-old shoots is inhibited by chloramphenicol, actinomycin D, and puromycin suggesting that protein synthesis is required. In 4-day-old shoots, actinomycin D and puromycin do not alter the kinetics of destruction while chloramphenicol partially inhibits the process. Thus, the inhibitors have a specific effect on the induction of the destruction mechanism but not its subsequent operation.