Cs2SO4 Density Gradients Research Articles

Nuclear and polyribosomal ribonucleoprotein particles containing poly (A) in rat liver cells.

Poly(A) containing ribonucleoprotein particles were prepared from rat liver nuclei and polyribosomes. The particles have sedimentation coefficients of 14 S and 9 S, respectively. In CS2SO4 density gradients the particles banded at densities of 1.28-1.29 g cm-3. Both nuclear and polyribosomal poly(A)-RNP contain in addition to some minor polypeptides, two main polypeptides having molecular weights of 63 000 and 90 000 dalton, respectively indistinguishable from each other according to their electrophoretic mobilities.

Isolation of L-cell messenger RNA which lacks poly(adenylate).

It has been found that centrifugation of (ethylenediamine)tetraacetic acid dissociated L-cell polyribosomes at 25 degrees C through preformed CS2SO4 density gradients containing 15% dimethyl sulfoxide resolves them into two distinct classes of particles that contain ribosomal and messenger RNA, respectively. Ribosomal RNA bands at a higher density than mRNA, presumably because it is more extensively stripped of protein by the action of CS2SO4 than mRNA. Undergraded RNA can be recovered from the gradients by trapping the particles on nitrocellulose filters and then eluting at 60 degrees C with a solution containing formamide and sodium dodecyl sulfate. When cells were labeled with [3H]adenosine, either in the presence or absence of a dose of actinomycin D which selectively inhibits rRNA synthesis, and their polyribosomes centrifuged through CS2SO4 density gradients, a large amount of the radioactivity recovered from the messenger-ribonucleoprotein region of the gradient had the size distribution of mRNA, but lacked poly(adenylate) (poly (A)). This material constituted 29-31% of the total mRNA. Histone mRNA was the most prominent component of poly(A)-lacking mRNA. However, 50-65% of the poly(A)-lacking mRNA was of larger size than histone mRNA.

The organization of repeated DNA sequences in the human genome.

The arrangement of repetitive and non-repetitive DNA sequences was studied in the human genome. By Ag+-Cs2SO4 density gradient centrifugations of human DNA at different fragment size reannealed to different Cot values and c-RNA hybridization experiments, we have shown the presence of two repetitive DNA fractions, called fast and slow intermediate DNA, with different pattern of sequence organization. The fast intermediate DNA sequences (6% of the genome; CsCl density in renatured form: 1.703 g/ml) are in part clustered in fragments greater than 24,000 nucleotide pairs and in part in fragments ranging from 1,800 to 600 nucleotide pairs spaced with longer more complex sequences. The slow intermediate DNA sequences (30% of the genome; CsCl density in renatured form: 1.707 g/ml) appear to be finely interspersed with non-repetitive sequences. At a DNA fragment size of 600 nucleotide pairs only a third of the slow intermediate DNA sequences are free of unique sequences, while the other two thirds are still organized with unique sequences. It has also been shown that a great amount of the repetitive DNA sequence transcripts in heterogeneous nuclear RNA of HeLa cells are complementary to slow intermediate DNA sequences.

Protein composition of nuclear 14 S ribonucleoprotein particles containing poly (A).

Nuclear 14 S RNP particles containing poly (A) from Ehrlich ascites carcinoma cells and rat liver were purified by re-sedimentation in sucrose gradients, by Cs2SO4 density gradient centrifugation and by affinity chromatography on a poly (dT)-Sepharose column. Proteins of these RNP particles were electrophoresed in urea and SDS-polyacrylamide gels. RNP particles of ascites carcinoma cells contain two main bands having molecular weights of 51 000 and 69 000 daltons, respectively, and two or three minor components.

Fate of viral RNA of murine leukemia virus after infection.

[3H]Uridine-labeled Rauscher leukemia virus was used to infect mouse embryo fibroblasts. After the infected cells were separated into nuclear and cytoplasmic fractions nucleic acid was extracted by sodium dodecyl sulfate-phenol-chloroform treatment and analyzed by Cs2SO4 and sucrose density gradient centrifugation. Between 45 and 70 min after infection a transient and synchronized shift of the acid-insoluble radioactive peak toward the RNA-DNA hybrid region occurred in both the nuclear and cytoplasmic fractions. The density of the cytoplasmic hybrid shifted to 1.56 g/ml (RNA equals about 50%), while the sedimentation rate decreased from 36 S to 14 S; however, the density of the nuclear hybrid shifted to 1.58-1.48 g/ml (RNA equals 57-17%, respectively), while its sedimentation rate remained about 65 S. The hybrids in both the nuclear and the cytoplasmic fractions still showed hybrid density after heat denaturation. The processes of the early stages of RNA tumor virus infection are discussed with regard to the functions of viral RNA-dependent DNA polymerase (reverse transcriptase) and a possible integration of viral genetic information into the host chromosome.

Homology between human breast tumour RNA and mouse mammary tumour virus genome.

SEVERAL studies have provided indirect evidence linking particles related to mouse mammary tumour virus (MuMTV) with human breast cancer1–3. In addition to morphological and biochemical similarities between human milk particles and known oncornaviruses shown in several laboratories4, Axel et al.5 demonstrated partial homology between MuMTV genome and polysomal RNA isolated from human breast tumour by molecular hybridisation experiments. Using Cs2SO4 density gradient analysis they showed that 67% out of 29 tumours tested had detectable amounts of MuMTV-related RNA. But because of intrinsic problems involved in Cs2SO4 gradient analysis it is not possible to determine hybridisation quantitatively. In this paper we describe hybridisation experiments using MuMTV-specific DNA probes and human tumour RNA under stringent conditions.

Synthesis of herpes simplex virus type 1 (HSV-1) DNA in isolated nuclei. II. Covalent linkage of RNA to nascent viral DNA.

Nascent HSV-1 DNA synthesized in vitro by isolated nuclei of infected rat embryo fibroblasts was found to be covalently linked to stretches of ribonucleotides. The nature of ribo- and deoxyribonucleotides at the RNA-DNA joint was investigated by assaying for the transfer of [32P]-radioisotope from an [α-32P]-deoxyribonucleotide to a 2’(3’)-ribonucleotide after alkali hydrolysis of the nascent HSV-1 DNA. All four common ribo-and deoxyribonucleotides were present at the RNA-DNA junction. To provide additional proof that the RNA pieces were linked to the newly synthesized DNA by a phosphodiester bond, the viral DNA was synthesized in the presence of labeled substrates for both DNA (α-32P-dCTP) and RNA (14C-GTP and14C-CTP). Analysis of the native and heat-denatured viral DNA-RNA copolymers by equilibrium centrifugation in CS2SO4 density gradients indicated that the [14C]-RNA did not dissociate upon denaturation and banded with the [³²P]-DNA.

5-Hydroxymethyluracil in the DNA of a dinoflagellate.

During the characterization of DNA from the dinoflagellate Gyrodinium cohnii, a large discrepancy was detected between the estimation of guanine + cytosine content from the buoyant density of the DNA in CsCl (56.1% G+C) and from the midpoint (T(m)) of its hyperchromicity induced by a thermal gradient (35.6% G+C). Composition analyses of (32)P-labeled nucleotides revealed an actual G+C content of 41.3%, and the presence of an unusual nucleotide amounting to about 37% of the expected thymidylate in unfractionated DNA-a feature that can explain the aberrant behavior of the DNA. The chromatographic properties of the unusual base and UV spectral analyses of the base and its corresponding nucleotide are consistent with its identification as hydroxymethyluracil. This base is not uniformly interspersed with thymine in the DNA. About 10% of Gyrodinium DNA is contributed by a fraction with low hydroxymethyluracil content, which behaves anomalously in Ag(+)-Cs(2)SO(4) density gradients but not in CsCl.

Wheat DNA: Study of the heavy satellite in Ag +–Cs 2SO 4 density gradient

Two DNA satellites can be observed in the wheat “Capitole” DNA centrifuged in an Ag+–Cs2SO4 density gradient. The heavy satellite (ϱ = 1.56 g/ml) is isolated and some of its molecular properties are analysed. It bands in neutral CsCl at the same density than the bulk DNA (ϱ = 1.702 g/ml) and renatures easily after thermal denaturation. The low molecular weight (300,000 daltons) and the structure of this satellite are discussed.

Modifications of genetic information in crown-gall tissue cultures

Summary Analysis of total DNA extracted from tumorous tissue cultures of Micotiana tabacum and Scorzonera hispanica by Ag+-Cs2SO4 density gradient centrifugation revealed a complex heavy satellite DNA at a density of 1.54 to 1.57 g/ml that is not found in corresponding healthy tissue; some molecular properties of this satellite are described. Implications of the presence of this reiterative fraction in crown-gall DNA are discussed.

Mechanism of carcinogenesis by RNA tumour viruses: the RNA-and DNA-dependent DNA polymerase activities of feline sarcoma virus.

SUMMARY In the absence of added template, the DNA polymerase of the feline sarcoma virus particle appeared to exhibit two-phase kinetics—a rapid reaction for about 4 min., followed by a slower reaction. Equilibrium sedimentation in Cs2SO4 density gradients showed the formation of RNA-DNA complexes within 12 sec., followed by the formation of DNA free of an RNA template. Virtually all of the DNA product formed at 4 min. and later was free of RNA template. About half of the 60 min. DNA product was double-stranded, since it was resistant to digestion by exonuclease I and eluted from hydroxyapatite columns at the salt concentration characteristic for duplex DNA. After denaturation, the 15, 30 and 60 min. DNA product sedimented at 6s and formed a broad band in CsCl gradients with an average buoyant density of about 1.725. Calf thymus DNA stimulated DNA polymerase activity of detergent-treated virus particle about 10- to 20-fold, indicating the presence of DNA-dependent as well as RNA-dependent DNA polymerase activity within the virus particle.

Virus interference by cellular double-stranded ribonucleic acid.

Ribonuclease-resistant ribonucleic acid (RNA) was isolated from uridine-labeled cultures of rabbit kidney, chicken embryo, and HeLa cells. This RNA, regardless of its source, was found to induce interference with virus growth in either rabbit kidney or chicken embryo cultures. Nuclease-treated cellular nucleic acids exhibited interference-inducing activity which eluted with a small fraction of RNA in the exclusion volume of a 6% agarose gel column. Besides resistance to ribonucleases, the interference inducer and RNA isolated from partially digested nucleic acids have in common two properties of double-stranded RNA: (i) similar sharp melting profiles were obtained for inducer and ribonuclease-resistant RNA, with T(m) dependent on NaCl concentration; (ii) ribonuclease-resistant inducer and RNA banded together in Cs(2)SO(4) density gradients at a density characteristic of known double-stranded RNA. After melting at low ionic strength, the labeled RNA shifted to a higher density and its capacity to inhibit virus replication was lost. Velocity sedimentation analysis of the cellular ribonuclease-resistant RNA indicated that the majority sedimented between 7 and 11S, but only RNA sedimenting at >==8 to 20S had a high specific activity of interference induction. Without prior ribonuclease treatment, the ribonuclease-resistant RNA can be precipitated with 2 m LiCl and thus appears to exist in purified cellular nucleic acids as part of molecular complexes with both single- and double-stranded regions of RNA. The biosynthesis of cellular double-stranded RNA is inhibited by actinomycin D.

Density gradient ultracentrifugation of rubella virus.

SummaryThe buoyant density of rubella virus HA was determined to be 1.23 in Cs2SO4. A density of 1.33 was obtained in CsCl. Rubella virus HA, either Tween-80. ether-treated or untreated, and infectivity could not be separated on sucrose by rate zonal or density gradient techniques. These activities also could not be separated on CsCl and Cs2SO4 density gradients. The banding density of rubella virus HA preparations was unaffected by the type of cell culture used for antigen production; neither the passage level nor the strain of virus altered the point at which rubella virus HA banded in CsCl.

Noncomplementarity in base sequences between the cohesive ends of coliphages 186 and lambda and the formation of interlocked rings between the two DNA's.

AbstractThe half molecules of 186 DNA have been isolated by the Hg(II)–Cs2SO4 density gradient centrifugal ion technique. The buoyant densities of the two halves in CsCI at 25°C. are 1.713 and 1.709 g./cm.3, corresponding to GC contents of 54% and 50%, respectively. Similarly, 5‐bromouracil labeled λ DNA halves were separated. The isolation of the four DNA halves made it possible to test for homology in base sequences between the cohesive ends of λ and those of 186. There was no indication of any significant homology in base sequences between the cohesive ends of the two DNA's, as indicated by the absence of a band with intermediate buoyant density in CsCI when either half of 186 DNA was annealed with either half of 5‐bromouracil labeled λ DNA and then centrifuged. The lack of cohesion between the two DNA's made it possible to demonstrate unequivocally the formation of interlocked rings (catenanes) between the two DNA's. The existence of a dimeric catenane is evidenced by the formation of a species of intermediate buoyant density when 5‐bromouracil labeled λ DNA is cyclized in the presence of cyclic 186 DNA of a relatively high concentration. The molecular weight of one DNA relative to the other can be calculated from the position of the dimeric catenane in a density gradient by using the method of Baldwin. The result was in complete agreement with our previous measurements from the sedimentation coefficients and by electron microscopy. The probability of dimeric catenane formation when one DNA is cyclized in the presence of another DNA is discussed. The experimental results agree with the theoretical expectation.

Suppression of RNA precipitation during Cs2SO4 density gradient centrifugation

Suppression of RNA precipitation during Cs2SO4 density gradient centrifugation

Spectrophotometric, potentiometric, and density gradient ultracentrifugation studies of the binding of silver ion by DNA

AbstractThe equilibrium and the stoichiometry for the reversible complexing of silver ion by DNA have been studied by potentiometric titrations, proton release pH‐stat titrations, and by spectrophotometry. The complexing reactions involve primarily the purine and pyrimidine residues, not the phosphate groups. There are at least three types of binding (types I, II, and III), of which the first two have been intensively studied in this work. The sum of type I and type II binding saturates at one silver atom per two nucleotide residues. In the type I and type II reactions, zero and one proton, respectively, are displaced per silver ion bound. At pH 5.6, the reactions occur stepwise, type I being first, while at pH 8.0, they occur simultaneously. The silver ion binding curve is very sharp at pH 8, indicating a cooperative reaction. The strength of the binding increases with increasing GC content. Type I binding is more important for GC‐rich DNA's than for GC‐poor ones. Denatured DNA binds more strongly than does native DNA. The silver ion complexing reaction is chemically and biologically reversible. We propose that type II binding essentially involves the conversion of an \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm N} - {\rm H} \cdots {\rm N} $\end{document} hydrogen bond of a complementary base pair to an N—Ag—N bond. The nature of type I binding is less clear, but it may involve a π interaction with stacked bases. The buoyant density (ρ0) of DNA in a Cs2SO4 density gradient increases when the DNA reacts with silver ion. The buoyant density change is about 0.15 g./ml. for 0.5 silver bound per nucleotide. The large buoyant density changes and the selective nature of the complexing reaction make it possible to perform good separations between native and denatured DNA or between GC‐rich and GC‐poor native DNA's by density gradient centrifugation.