Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea.

1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.     

Studies on the effect of gamma radiation (from Cobalt 60 source) on storage life of white yam (Dioscorea rotundata var. efon) infected with Scutellonema bradys

Studies on the effect of gamma irradiation on yam tubers infected with Scutellonema bradys showed that dosages between 5 and 15 krad did not eliminate the nematodes completely, but suppressed sprouting and signs of deterioration in the tubers. Dosages between 20 and 30 krad eliminated 70–80 % of the nematode population in yam peels. The appearance and taste of yams treated with between 7·5 and 15 krad were generally acceptable to a panel of tasters.     

The morphogenesis of green buds in γ - irradiated leaf and stem expiants of “aurea” chlorophyll mutantNicotiana tabacum L

The formation of buds in leaf and stem expiants derived from “aurea” mutant regenerants ofNicotiana tabacum (cv. John Williams Broadleaf, mutation Sulfur) was inhibited by dosages of 1 to 5 krad of γ-irradiation. The extent of the inhibition was proportional to the dosage of irradiation. In some eases, the stem expiants formed buds even after the application of 10 krad. In addition to “aurea” buds, green buds were also formed in both explants after-the irradiation with the dosages of 1 to 2.5 krad which continued to grow even after the trans plantation into garden soil.     

INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase

RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase.     

Tissue Browning of Potato Tubers Induced by Gamma Irradiation

A considerable browning was observed especially in cortex tissue and along xylem of potato tubers harvested at Sakai in Osaka Prefecture, after irradiation with 10, 20 and 50 krad doses of cobalt-60 gamma rays. This phenomenon was accompanied by the marked increase in polyphenol content and peroxidase activity, and the transient increase in o-diphenol oxidase activity. Total reducing compounds in the tissue were also increased by gamma irradiation.

The browning phenomenon depended on the storage period from the harvest to gamma irradiation treatment. The browning and the transient increase in o-diphenol oxidase activity were completely suppressed in the case of tubers irradiated 3 months after harvest.

There was no significant change in α-amylase activity in all tubers tested.     

Influence of Gamma Radiation on the Transport of Indolyl-3-Acetic Acid in Bark Tissue from Apple Trees

The influence of gamma radiation on the polarity of IAA translocation was investigated. Pieces of apple tree shoots, taken from 3-year-old trees, were irradiated in a dark irradiation chamber BK-⁶⁰Co source, 10 kCi. Doses from 20 to 150 krad were given. Strips of bark were peeled off both irradiated and control shoots. The middle parts of the bark strips were placed on agar blocks loaded with IAA-1-¹⁴C. The radioactivity of basal and acropetal segments was determined after about 20 h and results expressed as polarity quotient. It has been shown that irradiation with 20 krad (200 Gy) decreases the polarity quotient remarkably (polarity quotient of control equals 7; for 20 krad—2.9; 100 krad—1.6). The decrease in polarity results from inhibition of the basipetal translocation of IAA. Irradiated tissue shows a decrease in IAA uptake. Possible mechanisms of gamma irradiation effects on polar translocation of IAA are discussed.     

Chromatin-directed Ribonucleic Acid Synthesis: A Comparison of Chromatins Isolated from Healthy, Avirulent Agrobacterium tumefaciens Inoculated, and Crown-Gall Tumor Tissues of Vicia faba

Chromatin was extracted from healthy, avirulent Agrobacterium tumefaciens inoculated, and crown-gall tumor Vicia faba internodes of the same age. Chromatin from crown-gall tissue produced 5 times more RNA per 100 micrograms of DNA than chromatin from the healthy tissue. When template availability was compared using chromatin with saturating amounts of Escherichia coli RNA polymerase, chromatin from crown-gall tissue had 36% more available template than the controls. In addition, when γ-³²P-ATP was incorporated into the RNA synthesizing reaction mixture, with saturating amounts of E. coli RNA polymerase, there were twice as many RNA chain starts in tumor as in control tissue.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

The effect of gamma radiation on breaking of dormancy in Phycomyces spores

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.     

Chromatin-bound DNA-dependent RNA polymarase in developing pea cotyledons

The chromatin-bound DNA-dependent RNA polymerase activity, and template availability to added homologous RNA polymerase, were determined for pea cotyledons which were allowed to develop in different environments. Both the maximum polymerase activity and the template availability were higher in cotyledons that were developing at the faster rate. The template availability was approximately constant within an environment throughout most of the development, but differed between environments. The extra DNA sythesised, that is that above the 2C level, during cytyledon development appeared to be more utilised for RNA synthesis in slowly developing cotyledons than in more rapidly developing ones.     

Induced production of invertase in sugar-beet root by γ-irradiation: Role of RNA

Summary Gamma-irradiation dosages between 100 and 200 krad greatly stimulate the development of invertase activity in sugar-beet tissue. However, exposure of tissue to 800 krad virtually eliminates the production of invertase. The production of the enzyme in control and irradiated tissue requires RNA and protein synthesis. Failure of 5-fluorouracil to inhibit the development of invertase implies that the synthesis of ribosomal RNA is not required for enzyme production. A close correlation between irradiation-stimulated methylation of sRNA and enzyme production is noted. We suggest that the synthesis or modification of some RNA required for the translation of masked invertase RNA is stimulated by -irradiation.This research was supported by a contract (C00-1313-30) from the U.S. Atomic Energy Commission. This is journal paper 4473 of the Purdue University Agriculture Experiment Station.     

Nuclear and Mitochondrial DNA Synthesis in Gamma Ray-Resistant and -Sensitive Slime Mold Amebas

Uptake of [methyl-³H]thymidine label from Escherichia coli 15T^- into the DNA of Dictyostelium discoideum has been measured in control and [⁶⁰Co]-gamma-irradiated cells of the resistant strain NC-4 (D₁₀, colony-forming survival = 300 krad) and two sensitive daughter strains, γs-18 (D₁₀ = 75 krad) and γs-13 (D₁₀ = 4 krad). Nuclear (n) and mitochondrial (m) DNA were resolved by isopycnic CsCl gradients. The uptake of label into n-DNA during the immediate postirradiation period was selectively inhibited by irradiation, compared with uptake into m-DNA. For all three strains, the gamma ray dose to reduce the uptake into n-DNA to 37% of the control during the first hour after irradiation was 3 krad, whereas for uptake into m-DNA it was 75 krad. After the initial dose- and strain-dependent lag, uptake into n-DNA resumed. γs-18 showed longer lags in n-DNA synthesis and cell division than did NC-4. γs-13 resumed n-DNA synthesis and cell division after slightly shorter lags than for NC-4. The early postlag uptake into n-DNA in this strain was almost at the control rate and was accompanied by division until the cell number had nearly doubled. The rate of label uptake then declined, division stopped, and gradual cell lysis ensued. The postdelay response of γs-13 was almost independent of dose in the range of 10-100 krad. The response of γs-18 in these and earlier experiments is consistent with the view-point that it is sensitive because of a decreased rate of repair of DNA damage. However, the basis for the sensitivity of γs-13 seems to be more complex. This strain undergoes a premature but short-lived burst of n-DNA synthesis and division for what appears to be about one round of replication. Replication then ceases, even at very low doses, leading to greatly reduced probability of survival.     

SOME EFFECTS OF GAMMA IRRADIATION ON SEEDS AND RHIZOMES OF MUSA

The tolerance of Musa balbisiana Colla seeds to gamma irradiation was found to be considerably greater than that of rhizomes of the parthenocarpic variety ‘Gros Michel': e.g., 11.8 krad reduced the germination of rhizomes 92% and of seeds approximately 15%. Intact seeds exposed to doses higher than 48 krad did not germinate in non-sterile soil, but, when scarified and cultured axenically after irradiation, seeds which received doses as high as 70 krad germinated. Embryos excised from seeds exposed to doses as high as 285 krad formed callus, indicating that not all metabolic processes were inhibited by these extremely high doses. There was considerable variation in radiation tolerance between seed lots which was not related to their age, moisture content, or pre-exposure viability. Germination of intact seeds appeared to be stimulated by doses of 3 or 9 krad. No lasting differences attributable to the level of irradiation were apparent in the development of seedlings derived from either intact or scarified seeds nor of plantlets derived from excised embryos. Conversely, there was a significant reduction, proportional to irradiation dose, in the growth of plants developing from rhizomes, emphasizing the greater radiation sensitivity of the vegetative propagule. The radiation tolerance of seed-borne microorganisms was considerably higher than that of the plant materials, indicating that gamma irradiation is not effective as a means of obtaining pathogen-free rhizomes or surface-sterilizing seeds of M. balbisiana.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.