Quantitative aspects of mercurial-agarose gel electrophoresis

Single-stranded nucleic acids are capable of extensive intramolecular base pairing as well as intermolecular aggregation. Consequently, electrophoretic studies of single-stranded nucleic acids are most effective when conducted under denaturing conditions. A number of techniques are available for nucleic acid denaturing gel electrophoresis (1–3). In this paper we describe certain quantitative features of one of these techniques, mercurial-agarose gel electrophoresis (4–7). Specifically, we address the questions of resolution and base composition dependence and we introduce a new mercurial for agarose gel electrophoresis, p-chloromercuriphenyl-sulfonic acid.In a previous publication we demonstrated that methylmercury was an effective denaturant in an agarose gel (4). The mechanism of denaturation is presumably the disruption of hydrogen bonding by the reversible binding of methylmercury to uridine and guanosine imino nitrogens. At saturating mercurial concentrations accurate molecular weights can be determined, free of conformation effects. The presence of methylmercury has no observable effect on the mechanical properties of the gel. Hence the denaturing power of the gel can be readily varied. The strength and rigidity of agarose gels make them considerably easier to handle than acrylamide gels, and the large pore size ensures a system compatible with high molecular weight RNA.     

Isolation and separation of nucleic acids from Streptomyces hydrogenans (author's transl)]

Different methods for homogenization of cells of Streptomyces hydrogenans, for extraction of nucleic acids and for fractionation of the RNA and DNA obtained were critically examined. The only way to prepare high molecular weight rapidly labelled RNA and polysomes was to grind freeze-dried cells together with kieselguhr with a mortar and pestle. The best results for extraction of nucleic acids from the cell homogenate were obtained in the presence of diethyl pyrocarbonate (diethyl oxydiformate), yielding nucleic acids of considerable purity in a minimal amount of time. The best resolution of extracted nucleic acids was achieved by electrophoresis in 2% agarose acrylamide gels. This technique proved that during the cell homogenization and extraction procedure the bulk of nucliec acids was not degraded to low molecular weight material. An improved device for the registration of the profile of the absorption after gel electrophoresis is described.     

GelStar nucleic acid gel stain: high sensitivity detection in gels.

GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.     

Mechanics of chromatin template activation. Physical evidence for destabilization of nucleoproteins by polyanions.

Thermal denaturation profiles were used to quantitate the destabilizing activity of five classes of polyanions in an effort to explain previously reported differences in activity among apparently similar polymers. This physical assay for polyanion action on chromatin is free of the biochemical complications associated with template assay systems. Most polyanions tested caused a thermal destabilization of the normal nucleohistone complexes of rat thymus chromatin and exposed free or weakly complexed DNA. Polyphosphates dissociated chromatin even when their net charge was as low as 6, and activity per unit of weight increased with chain length only for polymers containing less than about 20 phosphate units; pyrophosphate was inactive. Two polymers which had no influence on chromatin had a low negative charge density. Some, such as polycytidlate and denatured DNA, possessed a high charge but caused only minor changes in the shape of denaturation profiles and exposed little or no free DNA. The highly active nucleic acids, ribosomal RNA and polyguanylate, were distinguishable from inactive nucleic acids by having more secondary structure and potentially less steric hindrance for anionic interaction with chromatin. There was evidence that some polyanions dissociate certain histones before others. Although data obtained show that some histones may be redistributed from chromatin onto added double-stranded DNA in a medium containing urea, this does not appear to interfere significantly with the interpretation of typical thermal denaturation profiles of chromatin.     

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5–10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.     

Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form.

The double-stranded form of cucumber mosaic virus-associated RNA 5 has been purified and further characterized. Its molecular weight determined by sedimentation equilibrium is 2.15 . 10(5). The buoyant density calculated from its symmetrical distribution in Cs2SO4, following isopycnic ultracentrifugation, is 1.615 g/cm3. The sedimentation rate of double-stranded cucumber mosaic virus-associated RNA 5 is slightly greater than that of cucumber mosaic virus-associated RNA 5; its electrophoretic mobility in polyacrylamide gel (2.4%) is less than that of cucumber mosaic virus-associated RNA 5. By the above standards the double-stranded cucumber mosaic virus-associated RNA 5 preparations used were found to be nomogeneous in size as well as density. Thermal denaturation monitored by means of ultraviolet light absorption produced multitransitional denaturation profiles. The average melting temperature (Tm) was 88 degrees C in 0.1 x SSC. Monotransitional denaturation profiles and slightly higher Tm values were obtained when resistance against ribonuclease digestion was measured. These denaturation experiments and other propertied led to the conclusion that double-stranded cucumber mosaic virus-associated RNA 5 and the double-stranded form of peanut stunt virus-associated RNA 5 are small double-stranded nucleic acids with several homostable base-pair regions, characterized by distinct G + C contents and Tm values.     

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.     

Isolierung und Charakterisierung schnell markierter,hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with ³²P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.     

Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis.

Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific RNA within the cytoplasm of HeLa cells infected for various times has been estimated. At 170-min postinfection, there are 0.67 X 10(5) molecules of single-stranded poliovirus RNA per cell and at 230 min, the amount has increased to 3.7 X 10(5) molecules/cell. Poliovirus double-strnaded RNA reaches a maximum of 0.7 X 10(5) molecules/cell at 330 min after infection.     

Methods for recovering nucleic acid fragments from agarose gels

Agarose gel electrophoresis is a powerful technique for the separation of nucleic acids on the basis of their size and conformation. The development of methods to recover size-fractionated nucleic acids molecules from agarose gels has greatly facilitated recombinant DNA technologies. Although several methods for recovering DNA and RNA molecules have been developed during the past fifteen years, none of them has been universally accepted. In this review we describe, discuss and evaluate the most common procedures with which we have had experience. Our evaluation is based on the criteria of yield, purity, speed, simplicity and low cost. We have considered three different approaches to the problem of recovering nucleic acids: chemical gel dissolution, physical gel disruption and physical extrusion from intact gels.     

Hybridization of nucleic acids directly in agarose gels

Nucleic acids, both DNA and RNA, separated on agarose gels can be visualized by direct hybridization of the dried gel with appropriate radioactive probes. This method does not involve the transfer of the nucleic acid from the gel. The method requires less manipulation than other procedures; it is extremely rapid, sensitive, and inexpensive. These attributes make this procedure a valuable alternative or supplement to the commonly used methods for visualization by hybridization of nucleic acids separated on agarose gels.     

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.     

Temperature of acrylamide polymerization and electrophoretic mobilities of nucleic acids.

The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 × 10⁶ to 2.5 × 10⁶ depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.     

Recent studies of some RNAs and proteins in amoebae

Progress on various aspects of nucleic acids and protein synthesis in amoebae has been reviewed. The RNA molecules involved in the character changes seen after micro-injection of non-homologous cytoplasmic fractions have been isolated after polyacrylamide gel electrophoresis, and their approximate molecular weights calculated. Injection of these RNA molecules was shown to alter the response of recipient cells to growth in streptomycin and neomycin.The relative molecular weights of cytoplasmic ribosomal RNAs have been estimated using both aqueous and formamide gel electrophoresis. Some attempts to characterize the nuclear RNAs seen on aqueous polyacrylamide gels, and to evaluate this data with that published by other workers have been made. Results from assays of DNA- and RNA-directed DNA polymerase activity are considered in relation to those from other eukaryotes.Problems arising after attempts to use rabbit globin messenger RNA to direct globin synthesis in amoebae, and the possibilities of using minature gel systems and small cell numbers to identify proteins and RNAs after various experimental treatments are discussed.     

Analysis of the electrophoretic properties of double-stranded DNA and RNA in agarose gels at a finite voltage gradient

The electrophoretic mobilities of double-stranded (ds) DNAs and ds RNAs of various lenths, L, were measured in gels of 0.4–1.8% (w/v) agarose at a voltage gradient of 1.0 V/cm. Differences in the electrophoresis of ds DNA and ds RNA are presented and discussed. A general expression is derived that describes the electrophoretic mobility, M, of either type of ds nucleic acid as a function of the gel concentration and the nucleic acid length: M = M′₁(L/L₀)^?x ? M₂, where M′₁ and L₀ are constants, and x and M₂ depend on the agarose gel concentration. The results obtained by fitting our data with this equation are consistent with the mobilities of nucleic acids in a wide range of gel concentrations, including free electrophoresis in solution and electrophoresis in gles of high agarose concentration in which nuleic acids are expected to reptate through the gel matrix. Finally, various methods of plotting agarose gel electrophoresis data are discussed.     

Effects of abscisic acid on nucleic acid metabolism in maize coleoptiles

Summary Following treatment with ABA an inhibition of total RNA synthesis was observed after 30 hours. Total soluble ribonuclease activity did not change during the first 8 hours, after which an increase could be observed.Separation of nucleic acids with polyacrylamide gel electrophoresis indicated that synthesis of soluble RNA was less inhibited by ABA than synthesis of ribosomal RNA.Effects of 5-FU and ABA on ribosomal RNA precursor were investigated. It could be shown that 5-FU did not inhibit ribosomal precursor synthesis, but that ABA did so.     

A simple and fast electrophoretic method for elution of nucleic acids from gels

A very convenient electrophoretic procedure for DNA or RNA elution from agarose or polyacrylamide gels is described. The gel piece with nucleic acid to be eluted is contained in a dialysis bag filled with buffer and elution is carried out in a horizontal electrophoresis apparatus. The nucleic acid is recovered with a high yield and can be used, without prior treatment, in further enzymatic or chemical reactions. Results obtained with DNA are presented here.     

Purification of 5S RNA from Plant Leaves

A simple and rapid method for large scale preparation of 5S RNA from plant leaves is described. To begin, all nucleic acids were extracted from the leaves with a mixture of phenol-chloroform-n-butyl alcohol and extracting buffer. After precipitation of the high-molecular-weight nucleic acids from the crude extract with 2 M LiCl, the low, molecular-weight RNA in the supernatant (containing about 6% 5S RNA) could be separated by eleetrophoresis on denatured polyaerylamide gels. The band of 5S RNA was excised from the preparatory slab gel under UV light and then purified by eleetrophoretie elution. In our experiments, several mg pure 5S RNA was obtained from 100 g leaves in a single run which takes about 4 days. The purified final produet was pure as showing a single hand on denatured polyaerylamide gel and a typical UV absorption peak of nucleic acid. The sedimentation coefficient (S20w) of the product was 4.6 as determined by ultracentrifugation.     

Comparative study of the ribosomal RNA from Leishmania and Trypanosoma

The ribosomal RNA from several stocks of the genera Leishmania and Trypanosoma were studied by gel electrophoresis, sedimentation on sucrose density gradients and RNA/DNA hybridization experiments. Three major components were observed after electrophoresis in polyacrylamide gels (PAGE-SDS), the relative molecular masses being respectively: X1 = 0.83 megadaltons, X2 = 0.63 megadaltons and X3 = 0.54 megadaltons for Leishmania RNA; and X1 = 0.86 megaldaltons, X2 = 0.78 megadaltons, and X3 = 0.58 megadaltons for Trypanosoma RNA. Depending upon the isolation procedure, a fourth component, X0 = 1.2 megadaltons (26S), became evident. The later component was purified from Leishmania brasiliensis (Y) by centrifugation on a linear 15-30% sucrose density gradient. This component, after heat denaturation and PAGE-SDS, gave rise to two bands coinciding in molecular mass with those of X2 and X3, indicating that these components are part of the large ribosomal subunit whereas X1 belongs to the small one. The above mentioned differences in mobilities of components X1 and X2 between the two genera were no longer observed after electrophoresis in denaturing agarose-formaldehyde gels, suggesting secondary structural differences among these RNA species. Hybridization experiments with L. brasiliensis (Y) DNA showed that both RNA types compete equally well for the ribosomal sites in this DNA, and that L. brasiliensis (Y) rRNA recognizes the ribosomal sites in DNA of Trypanosoma cruzi (EP), thus indicating that no gross changes occurred in their nucleotide sequences during evolution.