Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.     

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of ³H-TTP into DNA.     

EARLY EVENTS IN LYMPHOCYTE TRANSFORMATION BY PHYTOHEMAGGLUTININ : I. DNA-Dependent RNA Polymerase Activities in Isolated Lymphocyte Nuclei

The DNA-dependent RNA polymerase activities of isolated nuclei from lymphocytes were examined after stimulation with phytohemagglutinin (PHA). The nuclear fraction was prepared with Mg⁺⁺ or Mn⁺⁺ to distinguish between polymerase I (nucleolar) and polymerase II (nucleoplasmic). Distinction between polymerases II and III was obtained by the addition of α-amanitin to the reaction mixture. The results indicated that within 15 min after exposure to PHA the activity of polymerase I increased. Polymerase II activity increased after 1 hr. The enhancement was linear for 6 hr and then leveled off for the subsequent 48 hr. Small increase in polymerase III activity was observed at 48 hr. Inhibition of protein synthesis at the time of exposure to PHA did not prevent the increase in activities during the initial 6 hr. These results imply that the initial increase in enzymatic activities is dependent upon preexisting polymerase molecules and/or factors.     

Simple rapid procedures for isolation of tobacco leaf nuclei

The present paper describes and compares methods for the isolation of tobacco leaf nuclei. In contrast to standard procedures involving a long preincubation, a rapid cold ether treatment was found to aid in the release of nuclei in high yield. Two simple and rapid purification procedures are described which avoid the use of an ultracentrifuge. Relatively pure nuclei could be obtained in high yield in approximately 2 hr using the shorter purification procedures. The isolated nuclei were of acceptable quality as determined by (1) their appearance in the electron microscope, (2) their RNA, DNA, and protein content, and (3) their high activity in the DNA-dependent RNA polymerase reaction. Furthermore such preparations retained high RNA polymerase activity after at least 3 months at −20°C.     

Internation of zinc ions with DNA-dependent RNA polymerases A,B and C isolated from calf thymus

Purified DNA-dependent RNA polymerases A, B and C isolated from calf thymus contain a significant amount of zinc. Atomic absorption spectroscopy revealed the presence of 6.7, 5.35 and 2.6–4.1 g-atoms of zinc per mole of polymerase A, B and C, respectively. These enzymes are inhibited by treatment with 1,10-phenanthroline at concentrations varying from 10^-5 to 10^-4 M. However, the addition of zinc ions does not restore fully the activity of 1,10-phenanthroline treated enzymes. Exogenous zinc ions reducein vitro an overall RNA synthesis catalysed by RNA polymerases from calf thymus. In addition to the sites which bind zinc in a specific and stoichiometric way these enzymes possess other classes of binding sites with high and low affinity. Occupancy by exogenous zinc of these additional binding sites inhibits polymerase activity.     

An endogenous RNA-synthesis-promoting oligopeptide from Cucurbita pepto var. patissonina

The effect of CPPTI (Cucurbita pepo patissonina trypsin inhibitor) on the growth of Cucurbita pepo var patissonina (White bush) was examined. Plants treated with CPPTI grew faster than untreated plants and similarly to those treated with gibberellic acid. Isolated cell nuclei from plants treated with CPPTI showed that of the three DNA-dependent RNA polymerases assayed, RNA polymerase I (EC2.7.7.6) activity was preferentially elevated.Abbreviations CPPTI Cucurbita pepo patissonina trypsin inhibitor - GA₃ gibberellic acid - IAA indole-3-acetic acid - kDa kilodalton - RP I DNA-dependent RNA polymerase I     

Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.     

1H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli

The ¹H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α₂σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α₂), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.     

Nucleolar localization of ‘template-bound’ RNA polymerase I in nuclei of Xenopus laevis blastulae

This study shows, by means of autoradiography, that RNA polymerase I activity is present in some of the nuclei isolated from Xenopus blastulae. This activity is localized in one or two nucleoli which have a diameter of at least 1.5 μm. The ratio between nucleolar and total nuclear grain counts allows a quantitative estimation of RNA polymerase I activity relative to total RNA polymerase activity at the blastula stage.     

Distribution of fluorescent α-amanitin (FAMA) during mitosis in cultured rat kangaroo (PtK1) cells

The mushroom toxin α-amanitin is known to possess a high affinity to eukaryotic RNA polymerase II (or B) [1–3]. To pursue the question where these enzymes are located during mitosis of cells, a fluorescent derivative of α-amanitin (FAMA) was prepared. The affinity of FAMA to RNA polymerase II is 18 times lower than that of α-amanitin which is, however, sufficient for bright staining of nuclei of interphase rat kangaroo (PtK1) cells. During mitosis a large part of the fluorescent stain was distributed over the cytoplasm, while the chromosomes were never found to be stained. An accumulation of the fluorescent toxin during metaphase was observed in the spindle, particularly in the centrioles. Fluorescence of the centrioles persists also during anaphase. It is concluded that during mitosis of PtK1 cells the RNA polymerase II is distributed in the cytoplasm rather than bound to chromosomes. The accumulation of fluorescent toxin in the spindle and centrioles may speculatively be explained by the presence of another protein with high affinity to amatoxins, which has recently been isolated from calf thymus by Brodner & Wieland [4].     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes

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 α-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a two-fold 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 two-fold. 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 polymerase 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.     

Polypeptides of DNA-dependent RNA polymerase of spinach chloroplasts: characterization by antibody-linked polymerase assay and determination of sites of synthesis

Using solid-phase `Sandwich' immunoassays we studied DNA-dependent RNA polymerase of spinach chloroplasts with regard to (i) polypeptide composition of the multimeric enzyme; (ii) immunological cross-reaction with Escherichia coli RNA polymerase; (iii) sites of synthesis of polymerase polypeptides. Our main results are as follows. (i) All polypeptides of isolated chloroplast RNA polymerase (150, 145, 110, 102, 80, 75 and 38 kd) are labeled by an antibody-linked polymerase assay (ALPA), i.e., they are immunologically related to subunits of the holoenzyme. On the other hand differences in the patterns of `ALPA-reactive' polypeptides of a crude RNA polymerase fraction and of the purified enzyme preparation indicate partial proteolytic degradation of polymerase polypeptides during purification. Thus the 80- and 75-kd polypeptides, which had been previously considered as true RNA polymerase polypeptides, probably result from partial proteolytic degradation. (ii) The 150- and 145-kd polypeptides show immunochemical similarities with the β and/orβ' subunits of E. coli RNA polymerase. (iii) Results from solidphase immunoassay of in vitro translated products of both chloroplast RNA and poly(A)⁺ (nuclear) RNA suggest that all chloroplast RNA polymerase polypeptides are coded for by the nucleus.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.