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 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.     

Isolation of DNA-free RNA, DNA, and proteins by cesium trifluoroacetate centrifugation

The ability to simultaneously isolate intact DNA-free RNA, genomic DNA, and proteins from a biological specimen can be useful in cloning genes and analyzing gene expression. Equilibrium density gradient centrifugation with CsCl is a useful tool for fractionating, quantitatively separating, and characterizing RNA, DNA, and the total quota of proteins, respectively, based on differences in their buoyant densities. In the present study we have reexamined the rarely used cesium salt, cesium trifluoroacetate, for the same purpose. A significant advantage of CsTFA lies in the fact that, unlike in CsCl, RNA can be recovered from a single, soluble fraction of the CsTFA gradient. Furthermore, unlike CsCl, CsTFA is freely soluble in ethanol so that co-precipitation of the salt in the recovered RNA upon alcohol precipitation does not take place. Hence, the RNA is recovered with minimum manipulations. The one-step separation of cellular macromolecule classes free of each other in small amount of starting materials provides a major advantage over other methods currently in use.     

Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography

Covalently closed circular DNA can be isolated rapidly from cell lysates in a two-step process. Hydroxylapatite chromatography to prepurify the plasmid DNA from contaminating protein and RNA is followed by a step gradient elution of covalently closed circular (CCC) plasmid DNA from an acridine yellow affinity column. This procedure results in CCC DNA of a purity comparable to that obtained from ethidium bromide-CsCl gradients without lengthy centrifugation and free of contaimination by intercalating dye. Up to 250 μg of CCC pBR 322 can be isolated from 500 ml of bacterial culture in 4–6 h.     

Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [³H]or [¹⁴C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10⁶. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

His1, an Archaeal Virus of the Fuselloviridae Family That Infects Haloarcula hispanica

A novel archaeal virus, His1, was isolated from hypersaline waters in southeastern Australia. It was lytic, grew only on Haloarcula hispanica (titers of up to 10¹¹ PFU/ml), and displayed a lemon-shaped morphology (74 by 44 nm) previously reported only for a virus of the extreme thermophiles (SSV1). The density of His1 was approximately 1.28 g/ml, similar to that of SSV1 (1.24 g/ml). Purified particles were resistant to low salt concentrations. The genome was linear, double-stranded DNA of 14.9 kb, similar to the genome of SSV1 (15.5 kb). Morphologically, this isolate clearly belongs to the recently proposed Fuselloviridae family of archaeal viruses. It is the first member of this family from the extremely halophilic archaea, and its host, H. hispanica, can be readily manipulated genetically.     

Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid.

Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.     

Purification of plasmid DNA by an improved electrophoretic protocol

Plasmid DNA from Escherichia coli was isolated by electroelution carried out in an agarose gel that contains an incorporated dialysis membrane. As the relative mobility of circular plasmid DNA to linear chromosomal DNA increases when the agarose concentration is decreased, we were able to purify plasmids of up to 50 kbp in 0.3% agarose gel in Tris acetate buffer yielding 10-60 g DNA ml bacterial culture.     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Transition of R factor NR1 in Proteus mirabilis: molecular structure and replication of NR1 deoxyribonucleic acid.

The structure of R factor NR1 DNA in Proteus mirabilis has been studied by using the techniques of CsCl density gradient centrifugation, sedimentation in neutral and alkaline sucrose gradients, and electron microscopy. It has been shown that the nontransitioned form of NR1 DNA isolated from P. mirabilis cultured in drug-free medium is a37-mum circular deoxyribonucleic acid (DNA) with a density of 1.712 g/ml in a neutral CsCl gradient. This circular molecule is a composite structure consisting of a 29-mum resistance transfer factor containing the tetracycline-resistance genes (RTF-TC) and an 8-mum r-determinants component conferring resistance to chloramphenicol (CM), streptomycin/spectinomycin, and the sulfonamides. There are one to two copies of NR1 per chromosome equivalent of DNA in exponential-phase cells cultured in Penassay broth. After growth of PM15/NR1 in medium containing 100 mug of CM per ml, the density of the NR1 DNA increased from 1.712 g/ml to approximately 1.718 g/ml and the proportion of NR1 DNA relative to the chromosome is amplified about 10-fold. The changes in R factor DNA structure which accompany this phenomenon (termed the transition) have been studied. DNA density profiles of the transitioned NR1 DNA consist of a 1.718 g/ml band which is skewed toward the less dense side. The transitioned NR1 DNA consists of molecules containing the RTF-TC element attached to multiple copies of r-determinants DNA (poly-r-determinant R factors) and multimeric and monomeric autonomous r-determinants structures. Poly-r-determinant R factors have a density intermediate between the basic composite structure (1.712 g/ml) and r-determinants DNA (1.718 g/ml). These species presumably account for the skewing of the 1.718-g/ml DNA band toward the less dense side. When transitioned cells are subsequently cultured in drug-free medium, poly-r-determinant R factors and autonomous poly-r-determinants undergo dissociation to form smaller structures containing fewer copies of r-determinants. This process continues until, after prolonged growth in drug-free medium the NR1 DNA returns to the nontransitioned state which consists of an RTF-TC and a single copy of r-determinants.     

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

ABSTRACT: BACKGROUND: HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. METHOD: A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. RESULTS: The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. CONCLUSIONS: When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3--4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.     

Satellite DNA sequences in the red kangaroo (Macropus rufus)

There is a complex pattern of satellite DNA sequences in M. rufus which are revealed by addition of Ag+ or dye (Hoechst 33258) to the DNA ink Cs2SO4 or CsCl equilibrium density gradients. Six satellite DNA fractions have been isolated; these have buoyant densities in neutral CsCl of 1.692, 1.704, 1.705, 1.707 (two), 1.710 and 1.712 g/ml compared with 1.696 g/ml for the main band DNA. Each satellite accounts for 1-3% of the DNA of the genome. The satellites are located in the centromeric heterochromatin of the chromosomes, in the nucleolar organizer region and in interstitial bands on some of the autosomes, each satellite having a unique distribution. Nucleic acid hybridization showed that six of the satellite sequences are also present in the genomes of the wallaroo and the red-necked wallaby, with sequence divergences of only 1-2% relative to the sequences in the red kangaroo.     

SOME PROPERTIES OF DNA FROM ZOOXANTHELLAE HARBORED BY AN ANEMONE ANTHOPLEURA ELEGANTISSIMA1

Native DNA from the zooxanthellae harbored by an anemone, Anthopleura elegantissima consists of 2 species resolvable by differences in bouyant density in CsCl. Nuclear DNA has a density of 1.695 g/ml. Chloroplast DNA bands at a density of 1.688 g/ml. A third species of DNA, also with a density of 1.695 g/ml in the native state, is presumably mitochondrial DNA. It was detected in heat-denatured preparations. Nuclear DNA contains, in addition to the 4 usual bases, some 5-methyl cytosine. The latter was absent from chloroplast fractions.     

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Summary Intermediates of DNA replication in the second half of the latent period after phage infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative froms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the genome. Replicating circles with a linear DNA tail longer than one unit length of genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.     

Characterization of Escherichia coli nucleoids released by osmotic shock

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ～42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.     

Determining particle density distribution of expanded bed adsorbents

This study presents an experimental approach to measure the density distribution of expanded bed adsorption (EBA) matrices. We report on the use of a series of solutions of caesium trifluoroacetate (CsTFA) of varying density spun in a laboratory centrifuge so as to separate representative matrix samples on the basis of bead density. Mass data was used to plot a decumulative density distribution for the matrix. By performing laser light scattering-based measurements on the same samples of matrix the variation in particle size with density was determined. Particle settling velocity distributions were then calculated using these data and compared with a settling velocity distribution calculated on the basis of an assumed constant bead density. The study demonstrates a reliable and simple method for the characterisation of matrix density distribution. For the case of the Streamline matrices tested the particle size distribution is constant with varying bead density. Bead densities varied from 1.5 to 2.1 g/cm3 in the CsTFA solutions. These were then adjusted using bead porosity to give a density range of 1.11-1.33 g/cm3 in aqueous buffer (assumed 1.0 g/cm3) The differences in resultant settling velocity distributions when based upon measured density distribution than when based upon an assumed mean density value were shown to be insignificant. This result confirms experimentally that an assumption of a single constant mean density for EBA particles is acceptable for hydrodynamic modelling and performance prediction purposes.     

Detection of RNA-dependent DNA polymerase activity in the Xiphophorus melanoma system.

DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA-dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose-density-gradient centrifugation. The RNA-dependent-DNA-polymerase-like enzymes could be separated from the DNA-dependent DNA polymerases by DEAE-cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCl. Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA-dependent DNA polymerase isolated from Rous sarcoma virus.