A comparative study of the protein components of ribonucleoprotein particles isolated from rat liver and hepatoma nuclei

To solve the mechanism for the complete cessation of DNA synthesis in Tetrahymena cells involved in the amino acid starvation, the nature of DNA polymerase activity was investigated in crude enzyme preparations or in toluene-permeabilized specimens. In crude enzyme preparations from growing cells, ³H-TTP incorporation into acid-insoluble products showed little dependency on exogenous DNA template, while incorporation increased markedly in the presence of ATP. These characteristics were very similar to those of replicative DNA synthesis in permeabilized Escherichia coli.Variations of DNA and RNA polymerase activities following transfer of exponentially growing Tetrahymena cells to amino acid-deprived medium showed that in the crude enzyme preparations DNA polymerase activity dropped sharply within 3 h after the transfer and practically no activity was detected thereafter, whereas RNA polymerase activity did not disappear in the same preparations. Such enzyme kinetics coincided well with the kinetics of in vivo synthesis of the corresponding nucleic acid.The cessation of DNA synthesis in the amino acid-starved cells may be due not to the activation of DNase or a soluble polymerase inhibitor, nor to the deficiency of each kind of deoxyribonucleoside triphosphate or magnesium ion or ATP generation system. It follows from this that the cessation of DNA polymerase activity in the starved cells may be due to the deficiency of DNA polymerase or its associated factor(s) as a reflection of short life-span of such a protein.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

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.     

Inhibition of calf thymus DNA polymerase α and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine

6-(p-n-Butylanilino)uracil and N²-(p-butylphenyl)guanine inhibited the activity of DNA polymerase α from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases β and γ) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5′-triphosphate and did not occur in the reaction of DNA polymerase α with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases α.     

The binding of polynucleotides to the DNA polymerase of avian myeloblastosis virus.

The interaction between avian myeloblastosis virus DNA polymerase and synthetic nucleic acids was studied by an adaptation of the membrane filter binding technique. Bacillus subtilis DNA was used as a substrate for the binding reaction and was retained on the filters in the presence of the viral polymerase. The polymerase activity was demonstrated to be retained on the filter in either the presence or absence of the bacterial DNA. Characterization of the polymerase-DNA interaction demonstrated a marked similarity to previous data regarding the binding of Escherichia coli DNA-dependent RNA polymerase to nucleic acids when studied using related techniques. In contrast, the association between methylated bovine serum albumin and the B. subtilis DNA was found to differ significantly in both reaction stoichiometry and stability. Synthetic polynucleotides were shown to inhibit the binding of the bacterial DNA to the viral DNA polymerase and poly 2′-fluoro-2′-deoxyuridylic acid was found to be the most potent inhibitor of this reaction. Results from the binding-inhibition studies correlated well with studies concerning the inhibition of enzyme activity and it is concluded that the inhibitory polynucleotides act by interfering with binding of nucleic acid template to the viral enzyme.     

RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg²⁺ with optima at ? 0.01 and 0.02 M MgCl₂, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl₂ concentrations. In the presence of 0.01 M MgCl₂ the optimum (NH₄)₂SO₄ concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [₃H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated ? 3 pmoles of [₃H]UMP/muml; DNA at 25 C for 15 min, and ? 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, α-amanitin (20 m?/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with α-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).     

DNA Polymerase Activity associated with Purified Kilham Rat Virus

RNA tumour viruses contain an enzyme which can transcribe DNA from an RNA template^1,2, an endonuclease and a DNA-dependent DNA polymerase activity^3,4. RNA polymerase has been reported in vaccinia virus^5,6, reovirus^7,8 and cytoplasmic polyhidrosis virus⁹. I wish to describe a DNA polymerase activity associated with a highly purified preparation of the parvovirus, Kilham rat virus (KRV), which is thus the first report of a DNA polymerase associated with a DNA virus. KRV, a small virus first isolated from a rat sarcoma¹⁰, is antigenically related to the H viruses isolated from human transplantable tumours¹¹. Those parvoviruses which have been characterized all contain single stranded DNA with molecular weights of 1.5 to 2.5 × 10⁶ (refs. 12,13 and 14).     

The interaction of RNA polymerase and DNA Effects on the helix-coil transition and light scattering

To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transitions of poly[d(A-T] bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA. The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized. This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points. The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized. Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions. Core enzyme, on the other hand, aggregates extensively with double-stranded DNA, and only under conditions of low salt (10 mM KCl), without Mg²⁺, or at high salt (300 mM KCl), with or without Mg²⁺, can this aggregation be eliminated. Core enzyme also does not aggregate in the presence of single-stranded DNA. These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase.     

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.     

Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase in the Immune Response

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 M column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of ³H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12–13 S was most active as a template. It was followed by a fraction corresponding to 6–7 S. Sucrose gradient analysis of the ³H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

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.