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1.
Summary Small stable RNAs in wild-type Neurospora crassa were investigated by analyzing the cell contents of long term 32Pi labeled cultures in thin slab polyacrylamide gels. Because of the rigid fungal cell wall and the potency of nucleases the degradation of RNA in opening the cells was rather extensive. Some of these degradation problems were circumvented by using a slime strain of N. crassa which lacks a rigid cell wall. Our findings show that N. crassa. like many other eukaryotes, contains a number of small stable RNA molecules. We also found that the ribosomal RNA, the so called 5.8S, migrates slower on polyacrylamide gels than the 6S RNA of E. coli, which contains 184 nucleotides. The relative migration of the molecules was not changed when the samples were denatured prior to electrophoresis. The mobility of the Neurospora rRNA molecule suggested a chain length of 220 nucleotides. Fingerprinting of a T1 ribonuclease digest indicated a chain length of 212 nucleotides. Because of the unusually large size of the so-called 5.8S rRNA we found it more appropriate to refer to this molecule as a 7S rRNA. It seems that the N. crassa 7S rRNA is the largest low molecular weight ribosomal RNA studied thus far.  相似文献   

2.
Temperature-dependent uv absorption spectroscopy has been used to investigate the salt dependence of the order–disorder transition for the pH 4.2 rA8 double helix in 100% aqueous buffer and in a series of organic/aqueous mixed solvents. Melting temperature, Tm, data were obtained for the transitions in the different solvents by analysis of the uv melting curves. For the pure aqueous buffer solvent, the melting temperature was found to exhibit a reduced salt dependence (?tm/? log Na+) when compared to the corresponding polymer. This reduction is explained in terms of end effects and is shown to be consistent with the theoretical treatments of oligoelectrolyte transitions developed by Record and Lohman [Biopolymers, 17 , 159–166 (1978)]. In the mixed solvents, the salt dependence of the melting temperature (?tm/? log Na+) is shown to exhibit a linear dependence on the bulk dielectric constant of the medium for all of the hydroxyl-containing solvents studied. Significantly, N,N-dimethylformamide demonstrated different behavior.  相似文献   

3.
The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College (C log P), and Rekker's (log PRekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log kw and 0. Log kw was obtained by extrapolation of log retention factor (k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. 0 values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log kw values. For the methanolic eluents linear regression provided satisfactory correlations (r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations (r>0.99) were obtained between log kw and log P data using methanolic eluents. In such eluents 0 values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the 0-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.  相似文献   

4.
A direct method for measuring the derivative of thermal transition profiles (ΔAT), first described by Pavlov and Lyubchenko [Biopolymers 17 , 795–798 (1978)], is applied to the secondary structure of several eukaryotic ribosomal and messenger RNAs. The method consists of generating an effective ΔT between a sample and reference cuvette by altering the Tm (midpoint denaturation temperature) of one of the solutions with respect to the other. This can be done by changing salt concentration, solvent, pH, or ligands. Scanning the two cuvettes by varying the wavelength at different temperatures permits detailed examination of the base composition of differentially melting domains. We report here the ΔAT profiles generated by monovalent ion concentration differences for a number of high-molecular-weight natural RNAs, as well as the synthetic polynucleotides poly(rA) and “random” poly[r(A,G,U,C)]. The 18S and 28S rRNAs from chick embryos exhibit a reproducible series of peaks in the ΔAT profiles at low salt with ΔT = 4K. The high-temperature transitions in 28S rRNA appear to contain G·C base pairs exclusively, in contrast to those in 18S rRNA or any natural mRNA. Each mRNA we have examined (bacteriophage MS2, globin mRNA from rabbit reticulocytes, and procollagen mRNA from chick embryos) exhibits a distinctive ΔAT profile in low salt. The stability of many of the transitions in each of the mRNAs is no greater than that of the secondary structure in random poly(A,G,U,C) in low salt. More than 50% of the base pairing in procollagen mRNA actually “melts” below the mean for the random copolymer, indicating that despite its high G·C content, this mRNA contains a secondary structure that is exceptionally low in stability.  相似文献   

5.
Isolation of nucleic acids from plants by differential solvent precipitation.   总被引:46,自引:0,他引:46  
The purification of nucleic acids from plant tissue is often made difficult by the presence of contaminating carbohydrate polymers and polyphenols. A procedure for the simultaneous isolation of DNA and translatable RNA from plants is described. The method removes most of the polysaccharides and polyphenols extracted with nucleic acids in a single step by taking advantage of differences in solubility of these compounds in the solvent 2-butoxyethanol. Stepwise addition of 2-butoxyethanol to phenol extracts of specific ionic strength precipitates nucleic acids largely free of contaminants. Subsequent separation of RNA from DNA by precipitation with LiCl was optimised to give a high recovery of translationally active RNA. Successful isolation of nucleic acids from strawberry (Fragaria X ananassa) receptacle, a particularly recalcitrant tissue, and from a range of tissues of other plant species demonstrates the general applicability of the method.  相似文献   

6.
The temperature dependence of the hydrolysis of p-nitrophenyl carboxylates with general formula H(CH2)nCOOC6H4NO2 catalyzed by alkaline mesentericopeptidase has been studied (n varying from 1 to 7, temperature range 2–30°C, pH 8.80, 5 vol% dimethylsulfoxide). The activation parameters of the deacylation step depend on the length of the hydrophobic side chain of the substrate molecule ( , , and decrease by 2.0 kcal/mol, 4.9 kcal/mol, and 10 eu, respectively, as the length of the acyl carbon chain increases from n = 1 to n = 4). The following criteria were applied to establish a chemical enthalpy-entropy compensation effect: (a) Exner's plot of log vs : (b) Petersen's plot of log, k/T vs 1/T; (c) Exner's statistical treatment in coordinates log k vs 1/T; (d) according to Krug et al. (ΔH vs ΔGThm). By use of all the above-mentioned criteria the existence of a chemical enthalpy-entropy compensation effect was proved with an isokinetic temperature β of about 470°K, which is significantly higher than the average experimental temperature.  相似文献   

7.
The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and 19F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (Tm) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the 19F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D2O vs H2O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. 19F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T1), 19F{1H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.  相似文献   

8.
This report deals with the quantitative extraction of total nucleic acid (TNA) containing undegraded RNA from the slime mold Physarum polycephalum. With the use of a three-step phenol extraction technique, approx. 95 % of the nucleic acid optical density and 90 % of the 3H-uridine incorporated radioactivity were routinely recovered in the extracts. With the use of this technique it was shown that (1) the TNA mg dry wt of the mold did not change throughout the mitotic cycle, even though the dry wt doubled; this indicates a continual net synthesis of nucleic acid throughout the cycle; (2) the relative proportions of the various nucleic acid components did not change significantly during the cycle and were found to be DNA, 6 %; rRNA, 82 %; and sRNA, 12 %; (3) RNA molecules with mol wts of 4.1 m and 1.9 m, which exhibit properties of rRNA precursors were found in plasmodia labeled for 20 min with 3H-uridine. Furthermore, there appears to be an RNA fraction, found only in nucleic acid preparations presumably enriched in nuclear RNA components, which is heat-labile, does not enter 2.6 % acrylamide gels during 4 h of electrophoresis, and has a uridine/methyl ratio different from the presumed rRNA precursors and mature rRNA.  相似文献   

9.
The temperature dependency of the partitioning of p-alkylphenols and p-halophenols has been determined between dimyristoyl phosphatidylcholine liposomes and 0.15 M NaCl. Partition coefficients increased as a function of temperature below the endothermic phase transition temperature (Tc) of the phospholipid but decreased above this temperature. The transfer process was found to be entropy-dominated below and enthalpy-dominated above the Tc, although large negative entropy changes were observed. Regular changes in the thermodynamic functions, partition coefficients and functional group free energies occurred as a function of the alkyl chain length or size of the halogen substituent below but not above the Tc. This has tentatively been attributed to increased phenol-phospholipid interaction at the higher temperatures. The partitioning of p-fluorophenol behaved in a manner expected of fluorinated compounds, yielding relatively low partition coefficients, but it produced an additional effect of markedly lowering the Tc of dimyristoyl phosphatidylcholine. Good correlations of the partition coefficients in liposomes with those in bulk organic solvents and with molecular size of the solute have been obtained.  相似文献   

10.
Abstract

Guanine-rich polynucleotides such as poly(dG), oligo(dG)12–18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)25 was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG)·poly(dC), and, of those of the RNA type, preferentially poly(rA)·poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.  相似文献   

11.
Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.  相似文献   

12.
The objectives of this work were to demonstrate the potential of a two-phase sequencing batch reactor in degrading xenobiotics and to evaluate the kinetic parameters leading to a mathematical model of the system. 4-Nitrophenol (4NP), a typical representative of substituted phenols, was selected as the target xenobiotic; this compound has never been remediated in a two-phase bioreactor before. Partition tests were conducted to determine the most appropriate partitioning solvent, and among the three investigated solvents (1-undecanol, 2-undecanone and oleyl alcohol), 2-undecanone was chosen because of its favourable partition coefficient and its negligible emulsion-forming tendencies. Moreover, the selected solvent showed satisfactory biocompatibility characteristics with respect to the biomass, with only minor effects on the intrinsic microbial kinetics. Kinetic tests were then performed in a sequencing batch reactor (2-l volume) operated in both conventional one- and two-phase configurations, with the two-phase system showing a significant improvement in the process kinetics in terms of reduced inhibition and increased maximum removal rate. The obtained kinetic parameters suggest that the two-phase sequencing batch system may find full-scale application, as the maximum removal rate k max (~3 mg 4NP mgVSS−1 day−1) is of the same order of magnitude of heterotrophic bacteria operating in wastewater treatment plants.  相似文献   

13.
Xu L  Lv J  Ling L  Wang P  Song P  Su R  Zhu G 《Analytical biochemistry》2011,419(2):309-316
Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.  相似文献   

14.
RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA)   总被引:1,自引:1,他引:0       下载免费PDF全文
Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA–RNA duplex (ΔTm per modification: +2°C). A cyclohexenyl A nucleotide adopts a 3′-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4′-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The Vmax and Km of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the kcat value is about 600 times lower in the case of CeNA:RNA.  相似文献   

15.
A Monte-Carlo method including long-range interactions is used to oligopeptide chains in random-coil state. The chains are composed of 4, 9, or 14 repeating units and are labeled with the luminopheres tyrosine or tryptophan. Interactions with a solvent (water) are taken into account in the calculations through modifications of the semiempirical potential-energy functions. The chains represent oligopeptides composed of hydrophobic or hydrophilic amino acid residues. Various properties relavent to the interpretaiton of nonradiative enrgy-transfer experiments, such as the average value of the orientation factor for dipole-dipole interaction of the luminophores, 〈k2〉, the distribution function of the distances between the luminophores f(rl), the efficiences of energy transfer in the static and dyamic averaging regimes, 〈Ts amnd 〈Td, as well as the fluorescence decay I(t) of the donor luminophore in various averaging conditions, are computed. It is shown that, for all chains considered, 〈k2〉 is not vary far form 0.67 and that 〈Ts and 〈Td have completely different values. Due to the small extent of correlation between the distances rl and the mutual orientations of the lumninophores, the decay kinetics 〈I(t)s corresponding to a static averaging regime can be expressed in terms of distribution functions f(rl). These results are in agrrement with those obtained previously for the unperturbed chain model.  相似文献   

16.
Summary Heating of the TMV replicative form (RF) above a certain temperature (T m)causes a sharp shift from RNase resistance to sensitivity. The T mwas determined at different salt concentrations and in the presence of formamide.The kinetics of the annealing reaction between TMV RNA and its complementary RNA was studied, and the rate constant was estimated. Under the chosen conditions which are appropriate for annealing, no dissociation of double-stranded TMV RNA was detected. The kinetic data permitted a maximum estimate of the equilibrium constant of the annealing (or dissociation) reaction.  相似文献   

17.
The nucleotide sequences of all three rRNA operons (rrnA, rrnB, and rrnC) of Sphingobium chungbukense DJ77 were determined. The three rrn operons have the same gene order (16S rRNA-tRNAIle-tRNAAla-23S rRNA-5S rRNA-tRNAfMet). The nucleotide sequences were identical over a 5,468 bp region spanning the 16S rRNA gene to the 5S rRNA gene. Variability was observed in the 5S rRNA-tRNAfMet spacer sequence of rrnB. The tRNAfMet gene sequences were identical except for two bases (T5794 and A5871 in rrnB, T5942 and A5956 in rrnA, but C5942 and G5956 in rrnC). Comparative sequence analyses of ribosomal RNA operons from DJ77 with those of the class Alphaproteobacteria, to which the genus Sphingobium belongs, reveal close evolutionary relationships with other members of the order Sphingomonadales.  相似文献   

18.
Ursula Seitz  Ulrich Seitz 《Planta》1972,106(2):141-148
Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×106. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.  相似文献   

19.
The incorporation of the cytokinin N6-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N6-benzyladenine-8-14C or N6-benzyladenine-8-14C: benzene-3H(G) has been examined. N6-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N6-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N6-benzyladenosine content of the RNA preparation. The distribution of N6-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N6-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T2 and phosphatase, or ribonuclease T2 and a 3′-nucleotidase. N6-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T2 or phosphatase alone or by successive treatment with ribonuclease T2 and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T1 and pancreatic ribonuclease converted the N6-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N6-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.  相似文献   

20.
A new moderately halophilic sulfate-reducing bacterium (strain H1T) was enriched and isolated from a wastewater digestor in Tunisia. Cells were curved, motile rods (2–3 x 0.5 μm). Strain H1T grew at temperatures between 22 and 43°C (optimum 35°C), and at pH between 5.0 and 9.2 (optimum 7.3–7.5). Strain H1T required salt for growth (1–45 g of NaCl/l), with an optimum at 20–30 g/l. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as terminal electron acceptors but not nitrate and nitrite. Strain H1T utilized lactate, pyruvate, succinate, fumarate, ethanol, and hydrogen (in the presence of acetate and CO2) as electron donors in the presence of sulfate as electron acceptor. The main end-products from lactate oxidation were acetate with H2 and CO2. The G + C content of the genomic DNA was 55%. The predominant fatty acids of strain H1T were C15:0 iso (38.8%), C16:0 (19%), and C14:0 iso 3OH (12.2%), and menaquinone MK-6 was the major respiratory quinone. Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain H1T was affiliated to the genus Desulfovibrio. On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain H1T is proposed to be assigned to a novel species of sulfate reducers of the genus Desulfovibrio, Desulfovibrio legallis sp. nov. (= DSM 19129T = CCUG 54389T).  相似文献   

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