Purification of mucin glycoproteins by density gradient centrifugation in cesium trifluoroacetate.

A procedure for the rapid isolation of mucin glycoprotein by density gradient centrifugation in cesium trifluoroacetate (CsTFA) is described. The separation of mixtures of rat tracheobronchial mucin, DNA, hyaluronic acid, and bovine serum albumin in CsTFA gradients was superior to that in cesium bromide gradients. Inclusion of guanidinium chloride or urea in the gradient had no influence on the separation obtained. The mucins isolated from sputum samples of cystic fibrosis patients by this procedure are largely free of nucleic acid, nonglycosylated proteins, and glycosaminoglycans. The results of the use of CsTFA gradient centrifugation for the isolation of mucin from extracts of bovine submaxillary gland are also presented. The CsTFA method is particularly suitable for the high-yield isolation of mucin from individual samples which are available in limited quantities.     

Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients

A procedure for the simultaneous banding of cellular DNA, RNA, and protein by centrifugation in cesium trifluoroacetate (CsTFA) gradients is described. Starting with homogenates of Day 11 rat embryos, this procedure was used to separate total DNA, RNA, and protein. Under the conditions used DNA banded at a peak density of 1.63 g/ml, RNA at a peak density of 1.83 g/ml, and protein at a peak density of 1.40 g/ml. Nucleic acids isolated from CsTFA gradients were judged to be protein free. RNA isolated by this method is apparently free of DNA contamination; however, DNA isolated by this method does contain some RNA (less than 5% contamination).     

Purification and molecular structure of RNA polymerase from influenza virus A/PR8

The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.     

Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation

A rapid method for simultaneously banding preparative amounts of RNA and DNA from Trichinella spiralis muscle larvae by isopycnic centrifugation in cesium trifluoroacetate (CsTFA) is described. Larvae were homogenized in guanidinium isothiocyanate and the DNA, RNA, glycogen, and denatured protein components were isopycnically separated without prior purification. This procedure resulted in the isolation of nucleic acids suitable for molecular biological application. Agarose gel electrophoresis of gradient fractions indicated the separation of undegraded RNA and DNA where total RNA was of sufficient purity to efficiently direct in vitro translation of parasite protein and total DNA was greater than 20 kb in size and sensitive to restriction endonuclease digestion. Oligo (dT)-purified poly(A)+ mRNA was 3.6% of total RNA with greater than 18% conversion to cDNA.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Digestion of chromosomal proteins in formaldehyde treated chromatin

Treatment of chromatin subunits (nucleosome monomers) with formaldehyde results in the formation of cross-links between DNA and histones and between histones and histones. Digestion of chromosomal proteins with proteinase K does not lower the protein/DNA weight ratio below 0.08 to 0.1 as determined by cesium chloride gradient centrifugation of the digestion product from formaldehyde-treated nucleosomes. In addition to proteinase K, formaldehyde-treated nucleosomes were tested for accessibility to trypsin and pronase. The CsCl gradient patterns show, that pronase digestion and proteinase K treatment yield similar results. Trypsin treatment of control and formaldehyde-treated nucleosomes shows, that the sites which are accessible for trypsin in native nucleosomes, are blocked after formaldehyde treatment. Analysis of the CsCl gradient peak fractions in polyacrylamide gels shows, that the reliability of DNA fragment size determinations depends on the completeness of deproteinization.     

Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to alpha-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and alpha-amanitin resistance.     

Rapid step-gradient purification of mitochondrial DNA

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5-end labeling, gel retention assays, and various types of hybridization.     

On the hydration of DNA. II. Base composition dependence of the net hydration of DNA

The dependence of the net hydration of DNA on its base composition has been measured by density gradient ultracentrifugation of three DNA's in a series of cesium and lithium salt solutions of different water activities. Extrapolation to zero water activity showed the dependence of the partial specific volume on base composition to be very small for CsDNA and aero for LiDNA. At least 99% of the dependence of buoyant density on base composition can be accounted for on the basis of a differential hydration, with a mole of adenine–thymine pairs binding about 2 moles more water than a mole of guanine–cytosine pairs in CsCl.     

Improvement of DNA preparation and of PCR cycling in RAPD analysis of marine macroalgæ

In order to avoid the effects of bacterial contamination and the excess of RNA and polysaccharides coming from the cell walls, algal DNA for PCR cycling in RAPD analysis was extracted from protoplasts and purified in a CsCl gradient. Results indicate that RAPD can be efficiently applied to marine algæ and useful to distinguish between different strains or between different species.     

Localization and inactivation of DNase activity inClostridium pasteurianum NRRL-B598

Summary Each of four cell fractions ofClostridium pasteurianum NRRL-B598 contained DNase. The culture supernatant contained 31.3% of the total cellular DNase activity, while the cell wash, cell-wall compartmentalized and intracellular fractions contained 27.8, 27.8 and 13.1%, respectively. This enzyme was thermoresistant and heat treatment at various stages of the plasmid isolation protocol reduced chromosomal smearing, but did not improve plasmid recovery. The use of a new density gradient material (cesium trifluoroacetate; CsTFA) known to strongly inactivate DNase was compared to standard isopycnic centrifugation with cesium chloride. The consistent recovery of a 2.3 MDa plasmid only in the presence of CsTFA suggests that DNase continues to be a problem during ultracentrifugation as well as throughout the cleared lysate protocol.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Optimizing Phytoplasma DNA purification for genome analysis.

Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.     

Studies on the Organisation of the Chicken Genome and its Expression during Myogenesis in vitro

DNA from the chicken genome was analysed both by isopycnic centrifugation in cesium salt density gradients and by reassociation analysis using hydroxyapatite (HAP) chromatography. Centrifugation in neutral CsCl revealed a single non-Gaussian band skewed toward the heavy side, but no discrete satellite components. In heavy metal (Ag⁺ or Hg⁺⁺)-Cs₂SO₄ gradients, 4–8 satellite bands were revealed, comprising 5–9% of the total DNA. Purification of the satellites and recentrifugation in neutral CsCl demonstrated that 80–90% of this DNA would band in the shoulder, with the remainder in the main band. These satellites can account at most for 30% of the heavy shoulder DNA, thus most of the heavy shoulder DNA must be of lower repetition frequencies.
Reassociation analyses of chicken DNA demonstrated that the complexity of the non-repetitive DNA is 9.49 × 10⁸ nucleotide pairs, equivalent to about 90% of the haploid genome. Repetitive DNA comprises only 8–10% of the genome and has the following composition, relative to total DNA: 3.7% intermediate repetitive, 1.9% highly repetitive, and 3.9%"zero-time binding" DNA. This unusually low repetitive DNA content may be related to the small genome size of chickens, relative to other vertebrates, and to the presence of many microchromosomes in the chicken karyotype.
Total cell RNA extracted from prefusion myoblasts, post-fusion myotubes, and myoblasts grown in BrdU was incubated in large excess with ³H-TdR labelled non-repetitive DNA and the resulting hybrids assayed by HAP chromatography. The amount of non-repetitive DNA represented in the RNA was found to increase from 7–8% in the myoblast stage to 10–11% in myotubes. An even smaller proportion, about 5%, is represented in the RNA of myoblasts prevented from differentiating by growth in BrdU.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide

Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.     

Small-scale purification of bacteriophage λ DNA by an airfuge centrifugation step in cesium chloride gradients

A rapid and efficient procedure for purifying bacteriophage λ DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent λ DNA (20–40 μg each). The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.     

Transcriptive complex: isolation by cesium sulfate-centrifugation

Formation of the “CsCl complex” (an RNA polymerase-DNA complex isolatable by equilibrium centrifugation in CsCl) was further shown to require the presence of more than two species of nucleoside triphosphate substrates and double-stranded DNA suggesting that the formation at least of a single phosphodiester bond renders the RNA polymerase-promoter DNA complex resistant to dissociation by such high concentration of CsCl. Growing RNA chains were still attached to RNA polymerase-DNA complex even after centrifugation whereas completed chains were recovered as released from the complex and banded at the position of free RNA in Cs₂SO₄ equilibrium centrifugation.