Multiple DNA polymerase activity solubilized from higher plant chromatin

DNA polymerase preparations solubilized from chromatin of unwashed and washed sugar beet storage tissue exhibit multiple activity peaks when separated by DEAE-Sephadex chromatography. Activity peaks isolated from unwashed and washed tissue required Mg²⁺, all four deoxynucleoside triphosphates and added DNA. Activity from peaks isolated from washed tissue was completely sensitive to pancreatic DNase and insensitive to RNase. Enzyme activity was increased in the tissue during the washing period. In addition, there was a drastic change in template specificity from single-stranded primer in unwashed tissue to double-stranded DNA in washed tissue.     

Effects of gamma irradiation on chromatin activity of sugar beet tissue

Chromatin-associated RNA polymerase activity increases during washing of sugar beet tissue to a maximum by 20 hours. This increase was inhibited by dosages of gamma irradiation between 50 and 400 krad. Template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, also increased with washing and was inhibited, although to a lesser extent, by the above irradiation dosages. Neither endogenous polymerase activity nor template availability was affected by high dosages (300 krad) in unwashed tissue. Exposure of tissue to irradiation (300 krad) at different times during a 20-hour washing period severely inhibited the development of RNA polymerase activity during the early stages of washing. The inhibition of template availability, however, was independent of time of irradiation. The data presented are discussed in relation to the mechanisms involved in the inhibitory effects of gamma irradiation on RNA production and subsequent protein synthesis.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Evidence for DNA Polymerase-α and -β Activity in Sugar Beet

DNA polymerase preparations from incubated sugar beet (Beta vulgaris) root tissue exhibited multiple activities following DEAE-cellulose chromatography and glycerol density gradient centrifugation (600-fold purification). The two major polymerase activities eluted from DEAE-cellulose differed in heat lability, pH and Mg²⁺ optima, and in response to ethanol, p-hydroxymercuribenzoate, and N-ethylmaleimide. Further purification by glycerol gradient centrifugation indicated the presence of a low molecular weight molecule (3.4 S), heterogeneous larger molecules (6.2, 6.8 S), and apparent aggregates (11–11.5 S). This investigation indicates similarities between sugar beet polymerases and the small (β) and large (α) molecular weight polymerases from mammalian systems.     

The Presence of a Ribonuclease of High Molecular Weight in Sugar Beet Storage Tissue

Soluble ribonuckasie activity in sliced root tissue or sugar beet (Beta vulgaris) decreased to 30% of initial levels during the first 15 hours of aeration in sterile phosphate buffer. Activity increased with continued aeration reaching a maximum at 30 hours (85% of initial levels). Ribonuclease activity was isolated from unaerated tissue and partially purified by precipitation with acid and ammonium sulfate and by Sephadex chromatography. Enzyme activity was linear with respect to enzyme and substrate concentrations. The enzyme exhibited a substrate preference for RNA with no activity in the presence of native or denatured DNA. Elution of the enzyme from a Sephadex G-150 column indicated a molecular weight of 155,000. This uniquely large ribonuclease had no phosphodiesterase activity, was unaffected by ethylenediamine tetraacetate, and was inhibited by increasing Nig²⁺ concentrations.     

RNA polymerase activity in isolated nuclei ofNicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg²⁺ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH₄)₂ SO₄ and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.     

The development of invertase activity in slices of the root of Beta vulgaris L. washed under aseptic conditions

1. When disks of root tissue from sugar or red beet (Beta vulgaris L.) are washed in running aerated tap water the sucrose contained in them disappears and glucose and fructose are formed. 2. Invertase activity in the disks has been measured by a polarimetric method. Freshly cut tissue has a very low activity, but a considerable increase occurs during the first 3–4 days of washing, the final activity being sufficient to hydrolyse the sucrose contained in the disk within a few hours. 3. Disks of red beet have been cut and shaken in water under aseptic conditions. Sucrose breakdown and invertase development still took place. Microbial contamination is therefore not responsible. 4. Trisaccharides that appear in sugar-beet disks during the washing process have been isolated and identified; their formation also suggests that a higher-plant invertase is acting. 5. The significance of these results is discussed in relation to protein synthesis in washed storage-tissue slices, and the occurrence of high invertase activity in growing plant cells.     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities.

Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca²⁺ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca²⁺ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca²⁺ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca²⁺ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

An improved method for the isolation of intact nuclei from southern armyworm (Spodoptera eridania) larval midgut

Intact nuclei, relatively free of cell debris, were prepared from armyworm midgut tissue by a new, improved procedure involving homogenization in the presence of 0.1% Triton X-100 and 1% polyvinylpyrrolidone followed by sucrose density gradient centrifugation. The nuclear fraction thus prepared was a suitable source for assay of RNA polymerase activity. Polymerase activity was dependent on the presence of all four ribonucleotide triphosphates, was stimulated by Mn²⁺ and Mg²⁺ and was inhibited by actinomycin D and α-amanitin.     

pH effects on the removal of Cu2+, Cd2+ and Pb2+ from aqueous solution by waste brewery biomass

An industrial strain of Saccharomyces cerevisiae collected from the waste of a brewing industry was used to remove lead, cadmium and copper from aqueous solutions (1?mm). Metal removal efficiency by using either biomass suspension directly diluted into the metal solutions or biomass previously incubated and washed in distilled water was compared. In all experiments with unwashed biomass a shift in the medium pH from 4.5 to a final value in the 7.0–8.0 range occurred. This pH increase was responsible for a metal precipitation effect associated to the metal biosorption. A very different pH profile was observed when washed biomass was used leading to different removal profiles for Cd²⁺ and Pb²⁺ and a similar one for Cu²⁺. In the absence of biomass, medium components and/or the excreted intracellular products proved to interfere in the metal removal and to be responsible for 80% Pb²⁺ precipitation, in the pH 4.5–5.0 range. To initial metal solution pH, leading to the lowest residual ion concentrations, after 96?h of contact with unwashed biomass and in the absence of pH adjustment, was 4.5–5.0. Continuous or stepwise adjustment of medium pH to this range during the process was unfavourable for metal removal, being the continuous adjustment the worst procedure. In this case, Cd²⁺ was not biosorbed and Cu²⁺ removal decreased from 76 to 33%. However, Pb²⁺ was always extensively removed (89%) and only slightly affected by pH control. The global results suggest different removal mechanisms for each cation. Cu²⁺ was removed by both metal sorption and precipitation, due to the pH shift that occurred during the process, while Cd²⁺ removal showed to be completely dependent of this pH shift. Pb²⁺ was totally and quickly removed, by precipitation, in the presence of the biomass suspension and at pH 4.5. Moreover, the biosorbent changes occurring during the process played an important role in the metal removal when non-viable microbial biomass is used.     

On the difference between washing-induced and fusicoccin-induced K+ uptake in maize root segments

A comparison between fusicoccin (FC)-induced and washing-induced increase of K⁺ uptake in maize root segments is reported.K⁺ uptake rate nearly doubles after 4 h of washing as compared to the rate in the unwashed tissue. The stimulation of K⁺ uptake by FC is the same (approx. 1.6 μmol × g fresh wt.⁻¹ × 30 min⁻¹) both in unwashed and in washed tissue.Washing-induced increase of K⁺ uptake is strongly inhibited if 10⁻² M KCl is present in the washing solution; on the contrary, FC-induced increase of K⁺ uptake seems independent of the presence of KCl in the medium during the washing period.The inhibition of protein synthesis completely prevents the stimulation of K⁺ uptake induced by washing, while it only partially inhibits the effect of FC on the same process.These results, taken as a whole, suggest that the activation of K⁺ uptake induced by washing and that induced by FC depend on two different mechanisms.     

Rna polymerase from opaque-2 and normal Zea mays endosperm

RNA polymerase from Opaque-2 and normal maize showed qualitative differences during endosperm development. DEAE-Sephadex column chromatography indicated the presence of one and three RNA polymerases respectively at 15 and 25 days post-pollination. The polymerases from Opaque-2 and normal endosperms at 15 days post-pollination showed considerable differences in Mn²⁺ optimum. The optimum Mn²⁺ for normal polymerase was ten times higher than for Opaque-2 polymerase. The polymerase activity from endosperms at 15 days post-pollination was due to nucleoplasmic RNA polymerase II.     

Development and Characteristics of Sodium-selective Transport in Red Beet

Slices of storage tissue of red beet (Beta vulgaris L.) washed for only 1 day in distilled water readily absorb K⁺ but lack a mechanism for rapid Na⁺ uptake. A Na⁺ transport mechanism develops if the tissue is washed for several days, and the tissue then excludes K⁺ during Na⁺ uptake.     

Sucrose translocation and storage in the sugar beet

Several physiological processes were studied during sugar beet root development to determine the cellular events that are temporally correlated with sucrose storage. The prestorage stage was characterized by a marked increase in root fresh weight and a low sucrose to glucose ratio. Carbon derived from ¹⁴C-sucrose accumulation was partitioned into protein and structural carbohydrate fractions and their amino acid, organic acid, and hexose precursors. The immature root contained high soluble acid invertase activity (V_max 20 micromoles per hour per milligram protein; K_m 2 to 3 millimolar) which disappeared prior to sucrose storage. Sucrose storage was characterized by carbon derived from ¹⁴C-sucrose uptake being partitioned into the sucrose fraction with little evidence of further metabolism. The onset of storage was accompanied by the appearance of sucrose synthetase activity (V_max 12 micromoles per hour per milligram protein; K_m 7 millimolar). Neither sucrose phosphate synthetase nor alkaline invertase activities were detected during beet development. Intact sugar beet plants (containing a 100-gram beet) exported 70% of the translocate to the beet, greater than 90% of which was retained as sucrose with little subsequent conversions.     

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [³H]uridine) and in vitro (either by incubation of tissue slices with [³H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg²⁺-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn²⁺-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.