α-Amanitin-sensitive DNA-dependent RNA polymerase I from cherry salmon (Oncorhynchus masou) liver nuclei

DNA-dependent RNA polymerases I and II were purified approximately 3900- and 13300 fold, respectively, from a sonicated nuclear extract of the cherry salmon liver by column chromatographies on DEAE-Sephadex, heparin-Sepharose and DNA-cellulose. The RNA polymerases were examined with respect to template-specificity, the effects of Mn²⁺, Mg²⁺ and ammonium sulfate, α-amanitin sensitivity. Results showed that the RNA polymerase I differed from other eukaryotic RNA polymerase I in α-amanitin sensitivity.     

Localization of DNA-dependent RNA polymerase activities in fixed human fibroblasts by autoradiography

The sites of activity of DNA-dependent RNA polymerases may be demonstrated in the cell nucleus by incubating ethanol/acetone fixed cells with ATP, GTP, CTP and ³H-UTP. The amount of ³H-UMP bound to the chromatin in an RNase-digestible, acid-insoluble form is determined by autoradiography. The activity of one polymerase lies in the nucleolus and is inhibited by 0.05–0.5 μg/ml actinomycin D, another is present in the nucleoplasm and is blocked by α-amanitin. A large increase in α-amanitin-sensitive polymerase activity may be stimulated by performing the enzyme assay in the presence of a high concentration of ammonium sulphate.     

DNA-dependent RNA polymerases from Schneider 2-L cells of Drosophila. I. Preliminary characterization

The DNA-dependent RNA polymerases of Schneider 2-L cells of Drosophila melanogaster are described. These cells contain five readily detectable forms of this enzyme, polymerases I_a, I_b, III_a, II, and III_b, which elute from DEAE-Sephadex at 0.08, 0.12, 0.15, 0.20, and 0.22 m ammonium sulfate, respectively. RNA polymerases III_a and III_b, which each constitute about 5–10% of the total RNA polymerase activity in Drosophila embryos, are found to constitute 30 and 10%, respectively, of the total polymerase activity in cultured cells. The two form III polymerases are further characterized by in vitro response to divalent cations and ionic strength, template utilization, and sensitivity to -amanitin. Verification of the class III designation of these two polymerases is provided by their sensitivity to only very high levels of -amanitin (50% inhibition at approximately 800 µg/ml), their 10-fold greater activity on poly[d(A–T)], and their elution from DEAE-cellulose at lower ionic strengths than from DEAE-Sephadex.This work was supported by the Natural Sciences and Engineering Research Council.     

DNA-dependent RNA polymerase activities during embryogenesis in the bug, Oncopeltus fasciatus

Using α-amanitin to inhibit polymerase II activity in intact nuclei from Oncopeltus embryos, it is demonstrated that there is no difference in relative amounts of α-amanitin-resistant (Form I) and α-amanitin-sensitive (Form II) polymerases at two stages of embryonic development (70 and 140 hr), although the total polymerase activity is considerably higher at the earlier stage. However the RNA made under these circumstances (presumably due to Form I activity) appears to be, as expected, largely ribosomal.When the RNA polymerase activities are solubilized and separated, there is a substantially higher level of Form I activity in 70-hr embryos over that in 140-hr embryos. It is suggested that this high level of polymerase activity is correlated directly with the high level of ribosomal RNA synthesis at this stage.     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Isolation and characterization of DNA-dependent RNA polymerase A, B and C from rat brain nuclei

Abstract— DNA-dependent RNA polymerase activities were solubilized from the brain nuclei of young rats. Six forms of RNA polymerases were distinguished on DEAE-Sephadex A-25 chromatography and designated A, BI, BII, CI, CII, and Oil by their sensitivities to α-amanitin. CII enzyme was shown to derive from CIII enzyme by serine-protease digestion. CI enzyme was also suggested to be a product of a proteolytic process. Using a DNA template, enzyme A was completely resistant to α-amanitin; BI and BII enzymes were equally sensitive to this toxin (50% inhibition at 0.006 μg/ml); while C enzymes showed intermediate sensitivity (50% inhibition at 30 μg/ml). When poly[d(A-T)] was used as a template, α-amanitin sensitivities were altered in A, CI, CII, and CIII enzymes without any change in the BII enzyme. CI, CII and CIII enzymes were greatly stimulated by poly[d(A-T)], whereas A and BII enzymes were only slightly stimulated. All six forms of RNA polymerases were extensively characterized with respect to their ammonium sulphate optima, effects of divalent metal ions, template requirements and pH optima, using DNA and poly[d(A-T)] as templates. The results show new findings in several properties and supply basic data for discussion and future studies on RNA metabolism of the brain.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Variations in the amounts of RNA polymerase forms I, II and III during preimplantation development in the mouse.

DNA-dependent RNA polymerase has been measured at various stages of preimplantation development in mouse embryos. The total RNA polymerase activity per embryo increases rapidly from the 8-cell stage to the blastocyst stage. Studies with low α-amanitin concentrations, which inhibit form II RNA polymerase, and high α-amanitin concentrations, which inhibit both form II and III RNA polymerases indicate that the relative proportions of the three forms change significantly during preimplantation development. The changes which occur in the types and levels of RNA polymerase appear to parallel corresponding changes in the synthesis of the major classes of RNA.     

依赖于DNA的RNA聚合酶与体外转录的研究 Ⅰ.酵母变异株20B-12 RNA聚合酶A和C的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素和磷酸纤维素层析等步骤,纯化依赖于DNA的RNA聚合酶A和C,得到聚丙烯酰胺凝胶电泳均一的条带。其中不含DNase、RNase 和蛋白酶活力,无内源DNA。测定了 RNA 聚合酶A和C对α-鹅膏荤碱的敏感性。酶A在α-鹅膏荤碱为400μg/ml时,活性受到抑制,而酶C在该浓度时,几乎不受抑制。(NH_4)_2SO_4对酶A的最适浓度为20mM,对酶C有二个最适浓度,分别为40mM和240mM。无二价金属离子Mn~(2+)或Mg~(2+),酶A和C几乎无活力。两种酶最适Mn~(2+)浓度均为2.5mM,Mg~(2+)浓度均为5mM。两种酶以热变性小牛胸腺DNA为模板,测活性均较天然小牛胸腺DNA为模板时高。     

Nuclear DNA-dependent RNA polymerases of Ehrlich ascites tumor cells: two discrete -amanitin-sensitive forms

DNA-dependent RNA polymerase was solubilized from nuclei of ascites tumor cells by the standard techniques of ultrasonic treatment in 0.3 M ammonium sulfate, salt fractionation, and dialysis. Three discrete forms of RNA polymerase (I, II, III) were separated on DEAE-Sephadex A-25. Forms II and III were inhibited by α-amanitin, but no form was sensitive to rifampicin. Each form was more active with Mn⁺⁺ than with Mg⁺⁺ ions, more active with denatured than with native calf thymus DNA, and differed from the others with respect to optimal concentrations of (NH₄)₂SO₄, Mn⁺⁺ ions and DNA.     

Enzyme patterns in mitochondria of eggs, liver, and skeletal muscle during larval development of Xenopus

The amebo-flagellate Naegleria gruberi contains three major RNA polymerase activities separable from whole cell extracts by DEAE-Sephadex column chromatography. One is resistant to the durg α-amanitin and the other two are sensitive to it. These separated activities were characterized and then examined during the differentiation of ameba to flagellate, which is dependent on RNA synthesis and which exhibits gross changes in RNA metabolism under the conditions employed. It was found that no detectable changes occurred qualitatively or quantitatively in these polymerases during differentiation. Care was taken to account for total enzyme activity in every fraction of the extraction procedure. It is concluded from the above observation that gross changes in RNA synthesis as well as differential gene activity can occur with absolutely no major fluctuations in the DNA-dependent RNA polymerases.     

Evidence for two α-amanitin-resistant RNA polymerases in vegetative amoebae of Dictyostelium discoideum

Three DNA-dependent RNA polymerases (EC 2.7.7.6), P-I, P-II and P-III, have been isolated from the sonicated nuclear extract of vegetative amoebae of $\text{Dictyostelium discoideum}$ by phosphocellulose chromatography. P-I was inhibited by α-amanitin, while P-II and P-III were not. Rifampicin did not prevent all the polymerase activities. These polymerases were more active in the presence of Mg²⁺ than Mn²⁺. P-III was reduced in the enzyme activity by being passed through DEAE-Sephadex column and not obtained from the nuclear extract of amoebae at the culmination stage during morphogenetic development.     

Evidence for variation in the number of functional gene copies at the AmaR locus in chinese hamster cell lines

The hypothesis of functional hemizygosity has been examined for the α-amanitin resistant (Ama^R, a codominant marker) locus in a series of Chinese hamster cell lines. Ama^R mutants were obtained from different cell lines, e.g., CHO, CHW, M3-1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to α-amanitin inhibition, only about 50% of the activity is highly resistant in Ama^R mutants of CHW and M3-1 cell lines. The remaining activity in the latter cell lines shows α-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.