Protein and Ribonucleic Acid Synthesis by Plant Mitochondrial Systems

The incorporation of ¹⁴C-labelled phenylalanine into proteins of the mitochondrial systems obtained from 48-h germinating seeds of Vigna sinensis (L.) Savi can be stimulated by polyuridylic acid [poly (U)] and depressed by rifampicin, which is, however, ineffective if poly (U) is allowed to interact with the incorporating system before the antibiotic has access to it. A system consisting of a mitochondrial S-100 fraction and ribosomes from the same source with other cofactors can bring about polymerization of phenylalanine. The incorporation of ¹⁴C-labelled uracil into RNA by the plant mitochondria is greatly dependent on the exogenous addition of adenine, guanine, cytosine and also on 5-phosphoribosyl-l-pyrophosphate (5-PRPP). It is greatly suppressed by rifampicin and ethidium bromide.     

Synthesis and Biophysical Studies of Bis‐Macrocyclic Cobalt/Copper(II) Complexes Having a Pyridine Spacer with CT DNA and 5′‐GMP

New bis‐macrocyclic complexes of Co^III, 1 , Ni^II, 2 , and Cu^II, 3 , containing pyridyl bridges between 13‐membered macrocyclic subunits, have been synthesized via an in situ one‐pot template condensation reaction (IOPTCR). The proposed structures of these new dinuclear complexes are consistent with the data obtained from elemental analysis, molar conductance, IR, EPR, UV/VIS, ¹H‐ and ¹³C‐NMR, and ESI‐MS. The complexes 2 and 3 possess square‐planar geometry with four secondary N‐atoms coordinated to the metal ion, while complex 1 reveals octahedral geometry in solution due to coordinated H₂O molecules. DNA‐Binding properties of the complexes 1 and 3 were investigated by absorption and emission titrations, cyclic voltammetry, and viscosity measurements. Complexes 1 and 3 are strong DNA binders with binding constants, K_b, of 1.64×10⁵ and 2.05×10⁵ M ^?1, respectively. Hyperchromism, decrease in emission intensity of DNA‐bound ethidium bromide (EB), and changes observed in the viscosity and cyclic voltammograms in the presence of added metal complexes reveals that the complexes bind to DNA predominantly by electrostatic attraction, substantiated by absorption titration with 5′‐GMP.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1_c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10⁵ and 3.7 × 10⁵). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1_c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1_m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10⁵. Electrophoresis separated 1_m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10⁵ and 1.28 × 10⁵). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1_c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1_m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1_c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1_m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1_c but not 1_m. Electrophoresis after dissociation indicates that 1_c and 1_m are both enzyme complexes. After dissociation, the 1_c complex but not that of 1_m could be reassociated. The ribonuclease of the 1_m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1_c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1_m complex being concerned with RNA catabolism.     

Visualization of Drug-Nucleic Acid Interactions at Atomic Resolution. VII. Structure of an Ethidium/Dinucleoside Monophosphate Crystalline Complex,Ethidium: Uridylyl(3′-5′)Adenosine

Abstract

Ethidium forms a crystalline complex with the dinucleoside monophosphate, uridylyl (3′-5′) adenosine (UpA). The complex crystallizes in the monoclinic space group P2, with unit cell dimensions, a = 13.704 Å, b = 31.674 Å, c = 15.131 Å β = 113.9°. This light atom structure has been solved to atomic resolution and refined by full matrix least squares to a residual of 0.12, using 3,034 observed-reflections. The asymmetric unit consists of two ethidium molecules, two UpA molecules and 19 solvent molecules, a total of 145 non-hydrogen atoms. The two UpA molecules are hydrogen-bonded together by Watson-Crick type base pairing. Base-pairs in this duplex are separated by 6.7 Å; this reflects intercalative binding by one of the ethidium molecules. The other ethidium molecule stacks on either side of the intercalated base-paired dinucleoside monophosphate, being related by a unit cell translation along the a axis.

The conformation of the sugar-phosphate backbone accompanying intercalation has been accurately determined in this analysis, and contains the mixed sugar-puckering pattern: C3′ endo (3′-5′) C2′ endo. This same structural feature has been observed in the ethidium-iodoUpA and ethidium-iodoCpG complexes, and exists in two additional structures containing ethidium-CpG. Taken together, these studies confirm our earlier sugar-puckering assignments and demonstrate that iodine covalently bound to the C5 position on uridine or cytosine does not alter the basic sugar-phosphate geometry or the mode of ethidium intercalation in these model studies. We have proposed this stereochemistry to explain the intercalation of ethidium (as well as other simple intercalators) into both DNA and into double-helical RNA, and discuss this aspect of our work further in this paper and in the accompanying papers.     

Direct action of ethidium bromide upon mitochondrial oxidative phosphorylation and morphology.

Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount P_i released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of P_i during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [¹⁴C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A₃ was found to be 1.4 × 10⁴M^?1 and number of binding sites was equal to 3.48 ± 1.08 × 10¹² molecules/nuclei. The antibiotic chromomycin A₃ enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A₃ also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.     

Mitochondrial DNA synthesis during fungal spore germination

Summary Germinating spores of the fungus Botryodiplodia theobromae incorporated guanine-8-C¹⁴ into both the nuclear DNA and mitochondrial DNA fractions. Ethidium bromide inhibited the synthesis of mitochondrial DNA without having a significant effect on nuclear DNA synthesis or on the rate and extent of spore germination. Rates of leucine and uracil incorporation and of oxygen uptake were not significantly affected by ethidium bromide until germination was nearly completed. Mitochondrial DNA synthesis is apparently not required for germination of the spores of B. theobromae but is probably essential to continued vegetative growth.Abbreviations DNA deoxyribonucleic acid - mit-DNA mitochondrial DNA - nuc-DNA nuclear DNA - RNA ribonucleic acid - EB ethidium bromide - Tris tris (hydroxymethyl)aminomethane Published with the approval of the Director as Paper No. 3331, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. Paper No. 7877, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities.

Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca²⁺ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca²⁺ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca²⁺ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca²⁺ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.     

Visualization of Drug-Nucleic Acid Interactions At Atomic Resolution. VIII. Structures of Two Ethidium/Dinucleoside Monophosphate Crystalline Complexes Containing Ethidium: Cytidylyl(3′-5′) Guanosine

Abstract

This paper describes two complexes containing ethidium and the dinucleoside monophosphate, cytidylyl(3′-5′)guanosine (CpG). Both crystals are monoclinic, space group P2₁, with unit cell dimensions as follows: modification 1: a = 13.64 Å, b = 32.16 Å, c - 14.93 Å, β = 114.8° and modification 2: a = 13.79 Å, b = 31.94 Å, c = 15.66 Å, β = 117.5°. Each structure has been solved to atomic resolution and refined by Fourier and least squares methods; the first has been refined to a residual of 0.187 on 1,903 reflections, while the second has been refined to a residual of 0.187 on 1,001 reflections. The asymmetric unit in both structures contains two ethidium molecules and two CpG molecules; the first structure has 30 water molecules (a total of 158 non-hydrogen atoms), while the second structure has 19 water molecules (a total of 147 non-hydrogen atoms). Both structures demonstrate intercalation of ethidium between base-paired CpG dimers. In addition, ethidium molecules stack on either side of the intercalated duplex, being related by a unit cell translation along the a axis.

The basic feature of the sugar-phosphate chains accompanying ethidium intercalation in both structures is: C3′ endo (3′-5′) C2′ endo. This mixed sugar-puckering pattern has been observed in all previous studies of ethidium intercalation and is a feature common to other drug-nucleic acid structural studies carried out in our laboratory. We discuss this further in this paper and in the accompanying papers.     

Molecular conformations and 8-CH exchange rates of purine ribo- and deoxyribonucleotides: investigation by Raman spectroscopy

The rate of deuterium exchange of the 8-CH group in a purine deoxyribonucleotide, is the same as the 8-CH exchange rate in the corresponding purine ribonucleotide, with the exception of 5′-nucleotides of guanine. The observed 20% slower rate of 8-CH exchange in 5′-dGMP versus 5′-rGMP, over the temperature range 50–80°C, are attributable to differences in molecular conformation, including differences in ring puckering of the furanose substituents. Minor differences in 8-CH exhange rates are observed between 5′-and cyclic (3′:5′)-deoxyribonucleotides of a given purine, which are similar to those observed previously between corresponding 5′- and cyclic ribonucleotides that have been attributed to the charge difference of their respective phosphate groups [Ferreira, S. A. & Thomas, G. J., Jr. (1981) J. Raman Spectrosc. 11 , 508–514]. The coupling of guanine and furanose ring structures in the 5′-nucleotides is also evident from the vibrational frequencies of the guanine ring, which are strongly dependent on the pucker of the attached furanose moiety. Raman difference spectroscopy clearly reveals the dependence of purine nucleotide spectra on sugar-ring pucker. In the case of GMP, the guanine characteristic ring breathing mode near 600–700 cm^?1 depends for its exact position and intensity on the proportion of C3′-endo (668 cm^?1) and C2′-endo (682 cm^?1) conformers in equilibrium with one another. The Raman intensity ratio I(668)/I(682) is proposed as a measure of the conformer ratio C3′-endo/C2′-endo in 5′-dGMP with possible application also to nucleic acids. Among cyclic nucleotides, differences in spectra of deoxyribo- and ribo- forms also appear to be related to differences of molecular conformation.     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Conformations of cyclic 3',5'-nucleotides. Effect of the base on the synanti conformer distribution

The furanose and the phosphate rings of cyclic 3′,5′-nucleotides are locked in the ₄T³ and chair conformations respectively. The only variable which shows major conformational flexibility in these molecules is the rotation about the glycosyl bond which describes the orientation of the base relative to the sugar-phosphate bicyclic system. The glycosyl torsion angle has been analyzed for cyclic nucleotides with different purine and pyrimidine bases by use of conformational energy calculations. The results indicate that all the pyrimidine bases, U, T and C show a very strong energetic preference for the anti range of conformations. The calculations predict that among cyclic 3′,5′-purine nucleotides cyclic GMP and cyclic IMP favor the syn conformation to the anti by 95:5 and 70:30 respectively, while cyclic AMP shows a preference for the anti conformation to syn by 70:30. Thus the purines show a greater probability for the syn conformation than the pyrimidines in cyclic 3′,5′-nucleotides.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).