DNA-dependent RNA-polymerase II from human placental nuclei

The procedure for isolation and purification of RNA polymerase II from human placenta nuclei is described. The sensitivity of the enzyme to alpha-amanitin, bivalent cations, ionic strength and glycerol concentration in vitro has been studied. Eleven subunits of the RNA polymerase II molecule have been identified by gel electrophoresis. An optimal RNA polymerase II assay mixture has been developed.     

Thermosensitivity of nuclear RNA polymerase during the in vitro senescence of mouse fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro. The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture. Preheating nuclei increases the inhibition of their total RNA polymerase activity by alpha-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the alpha-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells. It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection

Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.     

Nucleic acid polymerizing enzymes in developing Strongylocentrotus franciscanus embryos

DNA-dependent RNA polymerase, DNA-Dependent DNA polymerase, and terminal riboadenylate transferase (TRT) activities have been measured after DEAE-Sephadex chromatography of whole cell extracts prepared from eggs and staged embryos of the urchin, Stronglyocentrotus franciscanus. Activity of each of these three polymerase classes is present in the egg, and the total activity per embryo is constant throughout embryogenesis to the pluteus stage (approximately 1000 cells). Thus the egg appears to contain sufficient DNA polymerase, RNA polymerase, and TRT TRT for embryogenesis. The increases in the synthesis of DNA, RNA and polyadenylated RNA tracts observed after fertilization must be due to the activation of the preexisting egg enzymes. Separation of the egg into nucleate and anucleate halves demonstrates that the RNA polymerases are not restricted to the egg nucleus. During development, the enzymes become progressively more associated with the cell nucleus. The egg extracts contain low activities (approximately 6% total) of RNA polymerase II as measured by sensitivity to alpha-amanitin. This is confirmed by resolution of the RNA polymerase forms I, II, and III by gradient sievorptive elution on DEAE-Sephadex. Later stage embryos contain more nearly equal activities of RNA polymerase, I, II, and III, although the total RNA polymerase activity per embryo is not changed. Additionally, two chromatographicallly distinct species of RNA polymerase III are detected, one of which is observed only in later stages. Thus interconversion of enzymes via addition of new subunits or coordinate synthesis and loss of enzyme species must occur.     

RNA polymerase activities in isolated nuclei of guinea pig seminal vesicle epithelium: influence of castration and androgen administration

Nuclei from seminal vesicle epithelium of adult guinea pigs were isolated in hypertonic sucrose solution. The incorporation of [3H]UTP by the isolated nuclei into acid-precipitable products was studied. Incorporation required ATP, GTP, CTP, UTP, and Mg+2. It was inhibited by addition of actinomycin D, deoxyribonuclease, or pyrophosphate to the reaction mixture. Thus, incorporation of [3H]UTP by isolated nuclei had the same characteristics that have been demonstrated for the reactions catalyzed by nuclear RNA polymerases. Using alpha-amanitin as a metabolic tool, we established concentrations of (NH4)2SO4. Mg+2, and nucleotides that give maximum assayable activities of nuclear RNA polymerases I and II. When the activities of polymerases I and II were measured in isolated seminal vesicle nuclei of guinea pigs that had been castrated 4 days earlier, a marked decrease in activities was found relative to control values (nuclei from intact animals). No further decrease was found 8 days after castration. Diminished accessibility to the nuclear DNA template and a decrease in the concentration of RNA polymerase molecules seemed to be responsible for the observed effects of castration on activities of RNA polymerases. An increase in ribonuclease activity did not seem to be responsible for the effects of castration. Activities of the enzymes did not change 2, 3, or 4 hours after intraperitoneal injection (2 mg/kg body weight) of each of five different androgens. Similarly, a single intraperitoneal injection of testosterone did not restore enzyme activity of polymerade I or II at any time during the first 24-hour period after hormone administration.     

An alpha-amanitin-resistant DNA-dependent RNA polymerase II from the fungus Aspergillus nidulans

An alpha-amanitin-resistant DNA-dependent RNA polymerase II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion-exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerase II can be purified over 6,000-fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg. The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two-dimensional gel electrophoresis using a non-denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170,000, 150,000, 33,000, 27,000, 24,000, 19,000, 18,000 and 16,000. Only one form of RNA polymerase II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerase II are clearly different from RNA polymerase I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin alpha-amanitin, even at concentrations up to 400 micrograms/ml, and appears to be unable to bind O-[14C]methyl-gamma-amanitin at a concentration of 10 micrograms/ml of the toxin. We conclude that the purified RNA polymerase from A. nidulans is a real, but exceptional, type of the class II RNA polymerases.     

In vivo effect of androgen and cycloheximide on the RNA synthesis in isolated nuclei of rat ventral prostates

Castration results in a rapid decrease in the activity of RNA polymerase I (or A) of isolated nuclei of rat prostates. The decrease was mainly ascribed to the diminution in the number of in vivo initiated RNA chains. The "free form" activity of the enzyme, however, which was estimated by the use of exogenous template and actinomycin D, increased 24 h after castration, then dropped rapidly. The administration of testosterone to castrated rats caused an increase in the activities of both RNA polymerases I and II (or B), which started 2 h and reached the maximum 12 h after the administration. No initial rise in RNA polymerase II activity was observed during the first 2 h. The administration of cycloheximide to normal rats caused a very rapid decrease of the activity of template-bound RNA polymerase I of isolated prostatic nuclei (t1/2=1.7 h), while the "free form" activity of the enzyme did not appreciably change until 3 h. The androgen-stimulated increase in the "engaged form" of the RNA polymerase I of isolated nuclei was completely abolished by the administration of cyclohexmide 60 min before killing. Based on the results obtained, the role of protein(s) with a rapid turn-over which is/are androgen-dependent and presumably participating in the control of preibosomal RNA synthesis is discussed.     

Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.     

RNA synthesis in alpha-amanitin-poisoned rats: prevention of recovery by inhibition of protein synthesis

The treatment with cycloheximide of rats previously poisoned with alpha-amanitin hinders the recovery of RNA synthesis observed in the liver of rats treated with alpha-amanitin alone. The recovery of RNA synthesis can be ascribed to the capability of poisoned rats to synthesize new RNA-polymerase II.     

Proceedings: Professional acculturation as an issue for training

1. Hydroxyindole-O-methyltransferase and DNA-dependent RNA polymerase activities were determined in the pineal gland removed from the ovariectomised rat and cultured under various experimental conditions. 2. The transferase activity declined very slowly during 24 h of incubation. 17beta-Oestradiol significantly increased the transferase activity within 2h after the addition, and the extent of increase was dose-dependent within the concentration range from 0.1 to 15 nM, being increased by 80% at 15 nM. Enhancement of the transferase activity by oestradiol was abolished not only by inhibitors of protein synthesis (cycloheximide and puromycin), but also by those of RNA synthesis (actinomycin D and alpha-amanitin). It was also blocked by clomiphene citrate, an agent which is known to inhibit the binding of steroid hormones to their respective receptors. 3. RNA polymerase activity (forms A and B) declined rapidly during the initial period of pineal culture. Oestradiol (15 nM) increased the RNA polymerase B activity by 50% within 2 h after the addition. The increase was dose-dependent within the concentration range from 0.1 to 15 nM, and was abolished by clomiphene citrate. 4. The possibility is suggested that the pineal is a target organ of oestradiol, and that the steroid-induced reaction sequence in the pineal conforms to that is known in other target organs.     

Synthesis of polyribonucleotide chains from the 3'-hydroxyl terminus of oligodeoxynucleotides by Escherichia coli primase

Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication. The sole known activity of primase is to catalyze synthesis of short RNA chains de novo. We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide. The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system. This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages. We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E. coli DNA polymerase III holoenzyme. The significance of this finding to primases generating multimeric length RNA is discussed.