Specific interference of metalaxyl with endogenous RNA polymerase activity in isolated nuclei fromPhytophthora megasperma f. sp.medicaginis

The systemic fungicide metalaxyl preferentially inhibits [³H]uridine incorporation into RNA by mycelium ofPhytophthora megasperma f. sp.medicaginis. Even at high concentrations of metalaxyl inhibition is not complete but circa 80%. Neither uptake of [³H]uridine nor its conversion into UTP is inhibited, indicating that interference with RNA synthesis takes place. Synthesis of RNA that lacks poly(A) sequences is more affected than that of poly(A)⁺ RNA. Metalaxyl has no effect on the activity of RNA polymerases present in mycelial extracts fromPhytophthora nor on that of polymerases I and II that have been partially purified with a procedure involving precipitation with polyethyleneimine, selective elution of RNA polymerases from the polyethyleneimine precipitate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. RNA polymerase II in mycelial extracts is half-maximally inhibited by α-amanitin at concentrations below 0.01 ¼g/ml. Both metalaxyl and α-amanitin inhibit endogenous RNA polymerase activity of isolated nuclei ofPhytophthora. According to their sensitivity to metalaxyl and α-amanitin, three types of endogenous activity can be distinguished: (a) an α-amanitin-sensitive type, the activity of which is stimulated by ammonium sulfate; (b) an α-amanitin-insensitive but metalaxyl-sensitive type; and (c) a type insensitive to both metalaxyl andα-amanitin. The first type of activity is characteristic of RNA polymerase II; the identity of the latter two remains to be elucidated. Metalaxyl andα-amanitin do not have any effect on free nuclear polymerases when assayed at a concentration of 50 mM ammonium sulfate with poly[d(A-T)] as exogeneously added template in the presence of actinomycin D to inhibit endogenous RNA polymerase activity. At 250 mM ammonium sulfate the free polymerase activity becomes α-amanitin sensitive but remains metalaxyl insensitive. Metalaxyl apparently inhibits RNA synthesis by specific interference with template-bound andα-amanitin-insensitive RNA polymerase activity. Endogenous polymerase activity of nuclei isolated from a metalaxyl-resistant mutant ofP. megasperma f. sp.medicaginis is not inhibited by metalaxyl, indicating that interference with RNA synthesis is the primary action of metalaxyl and that modification of the target site may lead to resistance.     

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.     

EHRLICH ASCITES TUMOR (EAT) CHALONE EFFECTS ON NASCENT DNA SYNTHESIS AND DNA POLYMERASE ALPHA AND BETA

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of ³H-thymidine (³H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50–200 μg/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude α and β-polymerase activities were inhibited. Crude DNA polymerase from C₃H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or ‘gapped’ DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting α- and β-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating that DNA ligase is not inhibited.     

Tryptophanyl-tRNA synthetase is found closely associated with and stimulates DNA polymerase α-like activity from wheat embryos

DNA polymerase α-like from wheat embryos is found to purify closely associated with a tryptophanyl-tRNA synthetase activity. No other aminoacyl-tRNA synthetases were present. A purified preparation of wheat tryptophanyl-tRNA synthetase free of polymerase activity was able to stimulate plant DNA polymerase of the α-like type, while the γ-like polymerase from wheat embryos was not affected by the enzyme. We have not been able to find a diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate binding activity associated to the polymerase-synthetase complex. We have also observed a specific inhibition by beef tRNA^Trp of DNA polymerase α-like activity, while other tRNAs will not change the enzyme activity.     

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.     

Diadenosine 5′,5‴-P1,P4-tetraphosphate- (Ap4A-) mediated stimulation of DNA synthesis in extracts from Xenopus laevis oocytes

Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) stimulates DNA synthesis in Xenopus laevis oocytes in the presence of activated DNA as template. Besides Ap₄A, other analogues such as Ap₃A, ATP and other derivatives are able to stimulate DNA polymerase activity. The effect of Ap₄A on DNA synthesis is observed with poly(dT) and poly(dT)-poly(dA) as templates, while no effect is found with poly(dA)(dT)_12–18 and poly(dC)(dG)_12–18. In the presence of a poly(dT) template, the oocyte extract is able to utilize Ap₄A as primer and to form a covalent bond between this dinucleotide and the nascent poly(dA) chain. An Ap₄A-binding protein present in the system has been purified and separated from DNA polymerase α-primase after phosphocellulose chromatography. After this separation, Ap₄A is no longer able to stimulate the polymerase activity, or to be utilized as primer by DNA polymerase α-primase.     

Relationship of ornithine decarboxylase to RNA polymerase I activity

Ornithine decarboxylase activity can be rapidly elevated 50- to 100-fold by the administration of methylxanthine derivatives such as methylisobutylxanthine. This elevation occurs just prior to the increase in RNA polymerase I activity. Inhibitors of RNA synthesis and of protein synthesis suggest that any alteration in the ornithine decarboxylase response results in a similar alteration in the level of α-amanitin-insensitive RNA polymerase. Addition of a partially purified ornithine decarboxylase preparation to the RNA polymerase assay increased both the initial rate of ³H-UTP incorporation and the length of time that the polymerase assay was linear. It is suggested that ornithine decarboxylase is the labile protein that modulates the level of RNA polymerase I.     

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).     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

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.     

Catecholamine binding to CNS adrenergic receptors

The properties of ³H-catecholamine binding to α- and β-adrenergic receptors in CNS are reviewed. ³H-epinephrine and ³H-norepinephrine label one class of α-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. ³H-epinephrine labels β₂, but not β₁, receptors in CNS, especially in bovine cerebellum. The same β-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na⁺-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

Detection of nicotinic cholinergic transmission in malapteruruselectricus electrop lax

Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, $\text{Malapterurus}$$\text{electricus}$, in order to determine if transmission was chemically mediated. There was no binding of [³H] acetylcholine, [³H] quinuclidinyl benzilate or [³H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [¹²⁵I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na⁺- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP⁺ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP⁺ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP⁺-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP⁺ exposure. We demonstrate that MPP⁺ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l^-/- Polh^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l^-/- Polh^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.     

Synthesis and assay of structural intermediates of crustacean pigment-dispersing hormones (α and β-PDH)

Summary

The two characterized crustacean pigment-dispersing hormones (α-PDH; β-PDH) are octadecapeptides which differ in primary structure at six positions. Assays for melanophore pigment-dispersing activity showed β-PDH to be 21-fold more potent than α-PDH. In an effort to explain the difference in potencies between the two PDHs, we synthesized and purified six analogs of α-PDH (Leu^4?, Leu^11?, Lys^13?, Asn^16?, Asp^17?, and Glu³, Leu^4? α-PDH) in which the amino acid residues of α-PDH were substituted with those of β-PDH. Four analogs (Leu^11?, Lys^13?, Asn^16?, and Asp^17? α-PDH) possessed melanophore-dispersing activity equivalent to α-PDH. Leu^4? α-PDH and Glu³, Leu^4? α-PDH were 2.4? and 4-fold more potent than α-PDH, respectively. Glu^3-α-PDH was 3.3-fold more potent than α-PDH (Jorenby et al., 1987). These results suggest that the 21-fold increase in activity of β-PDH over α-PDH is due to an interactive effect of two or more substitutions rather than from the product of the effects brought about by individual substitutions.