The effects of pH and salts on nucleic acid partitioning during phenol extraction

The partitioning of nucleic acids is sensitive to pH during phenol extraction. However, the exact effects of pH on phenol extraction had not been systematically investigated, and the mechanism of which were not fully elucidated. In this paper, we showed that the partitioning of nucleic acids was determined neither solely by the pH of the aqueous buffer being used, nor by the “pH of the phenol”; the latter is a completely wrong conception. We demonstrated that a key determinant for nucleic acid partitioning during phenol extraction was the equilibrated pH of the aqueous phase, which should be defined as the pH of phenol extraction. For example, when 50?mM NaAc-HAc buffer at pH of 3.47 was mixed with an equal volume of water-saturated phenol, the equilibrated pH of aqueous phase would be raised to ～3.84. At this pH, almost all of genomic DNA partitioned into the phenol phase, and genomic DNA-free total RNA was retained in the aqueous phase. Several salts were found affecting the partitioning of nucleic acids during phenol extraction in different manners. Based on these results, a low-cost and efficient method for genomic DNA-free total RNA extraction was developed.     

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T₁ ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.     

Factors Affecting the Activity of Phenolic Disinfectants

Low challenge phenol coefficient and high challenge use-dilution tests were made on a neutral cocoanut oil soap emulsion of o-phenylphenol and aqueous solutions of sodium o-phenylphenate prepared in the laboratory from the phenol using a stoichiometric amount of NaOH as well as with increasing amounts of excess NaOH. The phenol had considerably greater activity in both test methods when emulsified with the neutral soap than when converted to the phenate and dissolved in water. Use dilution test results against Salmonella choleraesuis with both the phenol and the phenate were within the range which would have been predicted from the Salmonella typhosa coefficient results employing the conventional conversion multiple of 20 to determine the maximal number of parts of water to which one part of germicide could be added. With the emulsified phenol this was also true where Staphylococcus aureus was employed in both procedures. With the aqueous solution of the phenate the maximal safe use-dilution by the phenol coefficient found for S. aureus and the same conventional conversion procedure was roughly five times higher than the maximal safe use-dilution found by the use-dilution method. Results with aqueous solutions of the phenate to which increasing amounts of excess NaOH were added showed no significant differences in the phenol coefficient method with either S. typhosa or S. aureus. In the use-dilution method, significant decreases in activity were found as the excess NaOH was increased to 4% with both S. choleraesuis and S. aureus. Although the pH values of aqueous solutions of the phenate were raised as the amount of free NaOH was increased, the decreases in pH observed as the dilution with water was increased were such that only small differences existed at the high critical killing dilutions found in the low challenge phenol coefficient method, whereas rather large differences existed at the lower critical killing dilutions in the high challenge use-dilution method.     

Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro

1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Application of dried sewage sludge as phenol biosorbent

The aim of this work was to determine the potential application of dried sewage sludge as a biosorbent for removing phenol from aqueous solution. Results showed that biosorption capacity was strongly influenced by the pH of the aqueous solution with an observed maximum phenol removal at pH around 6-8. Biosorption capacity increased when initial phenol concentration was increased to 110 mg/L but beyond this concentration, biosorption capacity decreased suggesting an inhibitory effect of phenol on biomass activity. Biosorption capacity decreased from 94 to 5 mg/g when biosorbent concentration was increased from 0.5 to 10 g/L suggesting a possible competitive effect of leachable heavy metals from the sludge. The effect of Cu2+ on biosorption capacity was also observed and the results confirmed that the phenol biosorption capacity decreased when concentration of Cu2+ in the sorption medium was increased up to 15 mg/L. Desorption of phenol using distilled deionized water was less than 2% suggesting a strong biosorption by the biomass.     

Simultaneous Extraction of RNA and DNA from Rat Liver with Cupric Sulfate

Homogenization of fresh rat livers in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate yielded both RNA and DNA in the aqueous phase after treatment vith phenol at 0–4°C. Effective deproteinization was achieved by three additional phenol treatments. Nucleic acids vere freed from Cu²⁺by repeated precipitation with ethanol in the presence of EDTA. The final yield was 6–7 mg/g of liver, of which about 20% was DNA and 11% tRNA. Physicochenical studies showed that pure tRNA, undegraded rRKA (30S, 18S) and native DNA (s°_{20, w} = 24.4S) were isolated by this procedure.     

Transfer RNA of a collagen producing tissue,chick-embryo calvaria

Transfer RNa was isolated from calvaria prepared from chick embryos incubated for 15–17 days. The chargeability of the unfractionated tRNA with ten amino acids tested was very similar to that of unfractionated tRNA from adult chicken liver when data were expressed on the basis of pmoles of amino acceptance per A₂₆₀ unit of tRNA. However, the relative amount of tRNA in calvaria is only about one-fourth the amount in liver. Analysis of individual species of tRNA by two-dimensional electrophoresis on polyacrylamide gels shows that there are fewer isoaccepting species of tRNA in calvaria than in liver.     

An improvement on labelled messenger RNA preparation for DNA-RNA hybridisation

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled lambda cI47 late m-RNA is prepared and hybridised to the r-strain lambda DNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3%. This low background m-RNA preparation is important especially in dealing with minor class m-RNA.     

Purification of human placenta phenylalanine, valine, methionine, glucine, and serine transfer ribonucleic acids.

By using column chromatography on varied media, the purification of several individual tRNAs from human placenta has been achieved. The crude human placenta tRNA was isolated using phenol extraction at pH 4.5 followed by DEAE-cellulose chromatography (B. Roe (1975), Nucleic Acids Res. 2, 21-42) and initially fractionated on BD-cellulose at neutral pH. Subsequent chromatography of the partially purified tRNA using high-speed, high-pressure liquid chromatography on RPC-5 and Aminex A-28 coupled with chromatography on BD-cellulose at acidic pH and on DEAE-Sephadex A-50 significantly shortened isolation time for milligram quantities of several pure tRNA species. Those tRNAs from human placenta obtained in a purity greater than 1.2 nmol/A260 unit are tRNAPhe, tRNAMet(i), tRNAVal(1a), tRNAVal(1b), and tRNAGly(1), while those obtained at purity of at least 0.8 nmol/A260 unit are tRNASer2 and tRNASer3. In addition, the use of Aminex A-28 as a chromatographic system for the isolation of tRNA is discussed.     

Fluorescent labeling of cell-free synthesized proteins with fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA

A simple and practical method for preparing fluorophore-conjugated methionylated tRNA necessary for tRNA-mediated fluorescent labeling of cell-free synthesized proteins was developed. Without complicated chromatographic purification and subsequent concentration, fluorophore-conjugated methionylated tRNA with higher purity and fluorescence concentration could be synthesized from in vitro transcribed tRNA instead of from a total tRNA mixture, which has been routinely used as a tRNA source. Although fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA was purified by simple phenol extraction following alcohol precipitation, it worked well in tRNA-mediated fluorescent labeling, yielding an improved signal-to-noise ratio and higher fluorescence intensity compared to the conventional total tRNA-based method. Based on its simplicity in the preparation of labeling agent with higher purity and fluorescence concentration, the developed method will accelerate the prevalence of fluorescence-based detection of cell-free synthesized proteins.     

An Improvement on Labelled Messenger RNA Preparation for DNA-RNA Hybridisation

Abstract

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled AcI47 late m-RNA is prepared and hybridised to the r-strain ADNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3% This low background m-RNA preparation is important especially in dealing with minor class m-RNA.     

Synthesis and characterization of a spin-labeled aminoacyl transfer ribonucleic acid

Unfractionated yeast transfer ribonucleic acid (tRNA) was reacted in aqueous acetone solution with the sulfhydryl spin-labeling reagnent, N-(l-oxyl-2,2,5,5-tetramethyl-3-Pyrrolidinyl)iodoacetamide. Whereas tRNA stripped of amino acids reacted only slowly, there were sites on tRNA charged with cysteine which combined rapidly with the reagent. The latter class of spin-label was quickly cleaved from the tRNA upon incubation in mildly alkaline solution (pH 8.0), suggesting that it was attached to the cysteinyl side chains. The paramagnetic resonance spectrum of column-purified spin-labeled cysteinyl tRNA showed that the spin-label was partially immobilized as a result of its interaction with the tRNA. When the tRNA was slowly heated, an abrupt increase occurred in the rotational mobility of the paramagnetic amino-acid side chain.     

The effect of ethylenediaminetetra-acetate on Pseudomonas alcaligenes and the composition of the bacterial cell wall

1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.     

Removal of chlorophenols from aquatic systems using the dried and dead fungus Pleurotus sajor caju

In this study, the potential use of the fungus Pleurotus sajor caju to remove phenols (i.e., phenol, o-chlorophenol, p-chlorophenol and 2,4,6-trichlorophenol) from aqueous solutions was evaluated. Biosorption of phenol or chlorophenols reached equilibrium in 4 h. The maximum adsorptions of phenol and chlorophenols onto the Pleurotus sajor caju were 0.95 mmol/g for phenol, 1.24 mmol/g for o-chlorophenol, 1.47 mmol/g for p-chlorophenol and 1.89 mmol/g for 2,4,6-trichlorophenol. The affinity order was as follows: 2,4,6-trichlorophenol> p-chlorophenol> o-chlorophenol>phenol. Phenol and chlorophenols bindings onto Pleurotus sajor caju were clearly pH dependent. The adsorption of phenol and chlorophenols increased with increasing pH. Desorption was achieved using methanol solution (30%, v/v). Pleurotus sajor caju biomass is suitable for reuse for more than five cycles without noticeable loss of adsorption capacity.     

Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.     

Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment

Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.