The Enrichment of Plasmid Dnas,in Bacterial Cell Lysates,Using an Alkaline-pH Procedure that Does Not Permanently Denature Them.

Abstract

The method described permits the enrichment of large (greater than or eoual to 54 kb), small (less than or equal to 2.1 kb), or intermediate sizes of plasmid DNAs. It is a modification of the plasmid enrichment method described by Currier and Nester (Anal. Biochem., 76, 431-441, 1976) in that it defers the alkali denaturation step until the pH and temperature can be controlled. This prevents permanent alkali denaturation of some plasmids. In general, total cellular nucleic acids are precipitated with either ethanol or isopropanol after lysates, in 3% w/v NaCl, are extracted with a phenol-chloroform mixture. The nucleic acids are then treated at an alkaline-pH (12.3-12.4), in a buffer, at 0°C, for a minimum time of 15 min. Denatured DNA, in 3% w/v NaCl, is removed with phenol. The RNA- containing, plasmid enriched fraction, is once again precipitated with either ethanol or isopropanol, dried in a vacuum, and redissolved. Samples are then digested with various restriction enzymes and/or examined directly on agarose gels after treatment with RNAase A.     

The use of ammonium acetate in the precipitation of ribonucleic acid.

Ammonium acetate (0.24M) in combination with cold 66% (v/v) ethanol quantitatively precipitated RNA from very dilute solutions (greater than or equal to mug/ml) after centrifugation for 2.5 X 10(6)g-min. There was also less co-precipitation of detergents with ammonium acetate than NaCl.     

A comparison of some procedures of salt extraction of nucleic acids from pollen. Elimination of interfering substances from extracts

Some methods were studied which use hot 10% NaCl solution for the extraction of both RNA and DNA from pollen. The raw salt extracts were precipitated with perchloric acid, trichloroacetic acid or ethanol and purified according to the described methods. The nucleic acid hydrolysates were obtained in several ways. In all the samples spectra in the UV-region were measured and the nucleic acid contents were determined according to the absorbance at 260 nm. In order to ascertain the extent of contaminants, the contents of phosphorus, saccharides and proteins were determined. It was found that by the methods studied it is possible to remove some impurities from extracts, but that the extractions of nucleic acids from pollen are not quite quantitative. A part of nucleic acids remained unextracted after the salt extraction in pollen, but it was possible to obtain it only by an additional extraction with 1 N perchloric acid at 75°C.     

A rapid method for preparation of bacterial plasmids

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.     

Flocculation onset in Saccharomyces cerevisiae: effect of ethanol, heat and osmotic stress

AIMS: To examine the effect of different stress conditions on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]; plasma membrane integrity was accessed using propidium iodide and the staining of the yeast cell wall was performed using calcofluor white M2R. Cells in exponential phase of growth were subjected to different stress conditions. The addition of 1%, 3% and 5% (v/v) ethanol, 1% and 3% (v/v) isopropanol or a brief heat shock (52 degrees C, 5 min), did not induce an early flocculation phenotype when compared with control cells. The addition of 10% (v/v) ethanol, a continuous mild heat-stress (37 degrees C) or an osmotic stress (0.5 or 1 mol l(-1) of NaCl) did not induce a flocculent phenotype. CONCLUSIONS: Flocculation seems not to be induced as a response to different chemical (ethanol and isopropanol) and physical (heat and osmotic) stress conditions. Conversely, osmotic and ethanol [10% (v/v)] stress, as well as a continuous mild heat shock (37 degrees C), have a negative impact on the phenotype expression of flocculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the control of yeast flocculation. This information might be useful to the brewing industry, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality, as well as in other biotechnological industries where flocculation can be used as a cell separation process.     

Combined solvent and water activity stresses on turgor regulation and membrane adaptation in Oceanimonas baumannii ATCC 700832

Oceanimonas baumannii ATCC 700832 is a Gram negative marine bacterium capable of utilising phenol as a sole carbon source. The ability of the bacterium to tolerate low water activity when utilising either succinate or phenol as a substrate in minimal medium was studied. The membrane lipid and protein composition showed two discreet adaptive phases as salinity increased. Firstly, when NaCl concentration was increased from 0.15% (w/v), the minimum at which growth was observed, to 1% NaCl (w/v), the ratio of zwitterionic to anionic phospholipids in the membrane increased significantly. At the same time the ratio of saturated to unsaturated fatty acids and the total membrane protein decreased significantly. The second phase was observed when salinity was increased from 1% to 7% NaCl (w/v) as the ratio of zwitterionic to anionic phospholipids decreased and membrane protein increased. However, the ratio of saturated to unsaturated fatty acids was unaffected. Salinity also affected the tolerance of cultures to elevated levels of phenol. Cultures grown in 0.15% NaCl (w/v) could tolerate 12 mM phenol, whereas in the presence of 1% NaCl (w/v) cultures continued to grow in up to 20 mM phenol and in 7% NaCl (w/v) cultures 8 mM phenol could be tolerated. Changes to the composition of the membrane phospholipids and fatty acids were also observed when phenol concentrations were at the maximum that could be tolerated. Under such conditions the ratio of zwitterionic to anionic phospholipids decreased twofold compared to cultures utilising 4 mM phenol as the substrate, in all salinities except in 7% NaCl (w/v) cultures, where there was no significant effect. The ratio of saturated to unsaturated fatty acids increased significantly in all salinities compared to cultures grown with 4 mM phenol. This revised version was published online in June 2006 with corrections to the Cover Date.     

Single-cell protein of Rhodotorula sp. Y-38 from ethanol, acetic acid and acetaldehyde

Summary Continuous cultivation of Rhodotorula sp. Y-38 was carried out on ethanol, acetic acid or acetaldehyde. At a feed concentration of 1.0 % (w/v) ethanol, the cell yield of 64 g/100 g ethanol and crude protein of 52 g/100 g biomass were obtained at D=0.5 h^-1. The respective value of the content of amino acids and nucleic acids was 42.6 and 9.4 g/100 g biomass. At 2.0 % (w/v) acetic acid, cell yield was found to be 50 g/100 g acetic acid at D=0.4 h^-1. The optimum dilution rate ranged between 0.3 and 0.4 h^-1. At 0.05 % (w/v) acetaldehyde, the maximum cell yield was obtained at D=0.14 h^-1.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

The circular dichroism and X-ray diffraction of DNA condensed from ethanolic solutions.

It is known that DNA in aqueous-ethanol solutions undergoes a B to A conformational change between 60% and 80% (w/w) ethanol. We have found that precipitates formed by adding salt to DNA in 60% and 80% ethanolic solutions can be very different. DNA precipitated from 60% ethanol forms a fine condensate that only slowly settles out of suspension and shows a characteristic differential scattering of circularly polarized light at long wavelengths. DNA precipitated from 80% ethanol forms a flocculent aggregate that exhibits the CD spectral features of the A conformation. Data from circular dichroism spectra of natural and synthetic nucleic acids and from X-ray diffraction patterns of the precipitates show that DNA molecules precipitated from 60% and 80% ethanol are, respectively, in the B and A conformation. Therefore, the different secondary conformations of DNA in ethanolic solutions are maintained during precipitation under these conditions. These results are of general importance for the preparation and study of condensed forms of DNA, since a relatively small change in the extent of dehydration can change the secondary conformation of DNA and markedly affect the character of a subsequent precipitate.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.     

Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K _D was performed using a novel distillation setup allowing stripping of ethanol at 50–65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55–65 °C) and exposure to gas–liquid and liquid–heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.     

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.     

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-I^Z solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-II^Z solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASH^(R)II Vector and Gigapack^(R)III XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Retention of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Plant growth, metabolism and adaptation in relation to stress conditions. XXVII. Can ascorbic acid modify the adverse effects of NaCl and mannitol on amino acids, nucleic acids and protein patterns in Vicia faba seedlings?

The adverse effects of either NaCl or mannitol on amino acids, protein patterns and nucleic acids in Vicia faba seeds were investigated. The exogenous addition of 4 mM ascorbic acid to the stressing media in which the broad bean seeds were germinated in combination with either the ionic (NaCl) or osmotic (mannitol) stressor induced significant protective changes in the total amount and in the relative composition of amino acids in general and in proline, glycine, glutamic, aspartic, alanine and serine in particular. It also induced changes in nucleic acids (RNA and DNA) content. These changes occurred throughout the entire period of the experiments (12 days). Separate administration of NaCl or mannitol enhanced the occurrence of particular novel proteins that were not detected in control bean seeds (water medium). Protein banding patterns of broad bean seedlings treated with NaCl or mannitol in combination with 4 mM ascorbic acid showed different de novo protein bands, with different molecular weights, at different stages of seedlings growth, with lower levels or a nearly complete absence of the major stress proteins. The pattern of changes for amino acids and nucleic acids and the range of protein bands extracted from the variously treated broad bean seedlings indicate a positive role of ascorbic acid in the alleviation of the damage effects induced by NaCl and mannitol. The importance of this role in the stress tolerance of broad beans is discussed.     

Activity and HPLC measured action pattern of Bacillus amyloliquefaciens α -amylase in water soluble organic solvents

The influence of two water miscible solvents (ethanol and isopropanol) on the activity of Bacillus amyloliquefaciens -amylase was studied.In ethanol-aqueous buffer (1:4, v/v) retained about 60% of the activity shown in water alone, both after l h hydrolysis. Isopropanol - aqueous buffer (1: 4,v/v) reduced the activity at 40%. The amount and the quality of produced oligosaccharides were effected by ethanol and isopropanol presence. In the mixture of produced oligosaccharides formed in the presence of the solvents only DP2, DP3 and DP6 were found. The disappearance of DP4, DP5 and DP7 which were formed in aqueous buffer suggest that a change in substrate affinity at the active centre is induced in the ethanol or isopropanol presence in buffer.Abbreviations DP degree of polymerization     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.