Change In Buoyant Density Research Articles

Buoyant density fractionation ofDrosophila melanogaster chromatin

Buoyant density fractionation of Drosophila melanogaster chromatin utilizing low molecular weight molecules such as actinomycin-D to induce changes in buoyant density has been investigated. Fractions of chromatin containing identifiable repeated DNA sequences could be isolated using actinomycin-D as the selective agent. Protein displacement from the chromatin complex was found to be a prerequisite for the observed buoyant density changes.

Endotoxin‐induced size change in bone marrow progenitors of granulocytes and macrophages

Injection of 5 mug endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.

A comparison of two populations of defective, interfering pseudorabies virus particles

Abstract The characteristics of two independently evolving populations of defective, interfering particles of pseudorabies virus passaged at high multiplicity of infection have been studied. Defective particles in both populations interfere with the growth of standard virus and contain DNA with a molecular weight similar to, or slightly smaller than, that of standard pseudorabies viral DNA. The two populations of defective virions differ with respect to the degree of reiteration, as well as with respect to the G + C content of their DNA. Interference by defective particles with the growth of standard virus is not correlated with a change in buoyant density of the DNA of these particles. Despite the difference in the composition of the DNA molecules of the two populations of defective virions, they behave similarly. Production of viral particles is reduced in cells coinfected with standard virus and either one of the two populations of defectives, as compared to cells infected with standard virus alone. Maturation of concatemeric nascent forms of viral DNA to unit size molecules is also delayed.

Buoyant and potentiometric titrations of synthetic polpeptides. II. Five copolypeptides and two nonionizable homopolypeptides in CsCl solutions.

AbstractThe buoyant density titrations of five ionizable copolypeptides in concentrated CsCl solutions have been determined. The results are used to formulate models for predicting the buoyant density titration behavior of copolypeptides and proteins using the previously reported homopolypeptide buoyant density titration curves. It was determined for these copolypeptides that the best predictive model must include not only the buoyant densities of the constituent amino acid residues and the relative composition, but also hydration and salt binding.Hydrations determined for the homopolypeptides are used in the copolypeptide predictive model. The hydrations of the neutral homopolypeptides were readily calculable since their buoyant densities are thermodynamically defined in terms of their partial specific volumes and hydrations. For the case of a charged macromolecule, an expression for the buoyant density as a function of the number and nature of the bound ions, its partial specific volume, and its relative hydration has also been available for some time. This heretofore intuitive relationship is now derived from thermodynamic principles and allows calculations of hydrations to charged macromolecules which bind either cations, anions, or both.The potentiometric titrations of three of the five copolypeptides in concentrated CsCl solutions were determined in order to study the effect of residue interaction and solvation effects on their ionization behavior. The potentiometric results are also combined directly with the buoyant density titration results to determine the correlation of the buoyant density with the degree of ionization. As in the cases of poly(Glu) and poly(His), the buoyant density of the copolypeptides changed linearily with the degree of ionization.The buoyant density titrations of two nonionizable homopolypeptides, poly(Gly) and poly(Ala), were determined in concentrated CsCl solutions. The buoyant density was found to increase with increasing pH, despite the fact that side chains do not contain ionizable groups. This is the first evidence from homopolypeptide or copolypeptide data that buoyant density changes can be observed from effects other than side‐chain ionizations.

The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA

1. The effect of pH on the buoyant density of the complexes of Ag + with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral Cs 2SO 4 density gradients, Ag + complexes with (G · C)-rich components are always denser than those with (A · T)-rich components, since (G · C)rich DNAs have a larger affinity for Ag + than (A · T)-rich DNAs and their complexes are denser than (A · T)-rich complexes. In alkaline ( pH > 9 ) Cs 2SO 4 gradients, the buoyant density of the Ag + · DNA complex is not a simple function of base composition. The Ag + affinity of (A · T)-rich DNA is larger than that of (G · C)-rich DNA but the density of a (G · C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag +. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides

The effect of Hg 2+ and Ag + on the buoyant density (ϱ) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) · poly(dT); poly[d(G-C)]; and poly(dG) · poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs 2SO 4 increased dramatically after complexing with Hg 2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg 2+ DNA-P ( r f) Hg 2+ raised the thermal transition temperature ( T m) of alternating polymers and lowered the T m of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered T m correlated with Hg 2+-induced strand separation of one homopolymer [poly(dA) · poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) · poly(dC)] complexed with Hg 2+. When Ag + was mixed with the polymers, the buoyant density of poly(dG) · poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The T m changes induced by Ag + were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg 2+/Cs 2SO 4 and Ag +/Cs 2SO 4 gradients.

Excision repair in Tetrahymena: events following refeeding of starved UV-irradiated cells.

Abstract—Starvation of early‐log‐phase Tetrahymena pyriformis in non‐nutrient phosphate buffer for 24 h results in a 40 per cent increase in cell number, as well as a complete cessation of DNA synthesis. Low levels of DNA synthesis are detectable between 1 and 2h after starved cells are transferred to a nutrient medium. Larger amounts of DNA synthesis are detected after the first 2 h of refeeding, and one round of replication is complete 4.5 h after refeeding. Damage, caused by sublethal doses of UV radiation (254 nm) administered just prior to refeeding, to the DNA of starved Tetrahymena appears to be corrected by an excision‐repair process after refeeding of starved, irradiated cells. Changes in buoyant density of DNA synthesized, rate of DNA synthesis, and the chromatographic distribution of photoproducts were investigated following refeeding of starved, irradiated cells. Excision repair begins 1 h after refeeding and appears to be essentially complete within 7 h. During this time, thymine dimers produced by irradiation are removed. Semiconservative DNA synthesis commences 2–3 h after the first appearance of excision repair. In addition, between 3 and 8 h after refeeding, the rate of DNA synthesis in irradiated, refed cultures is much lower than the rate of DNA synthesis in unirradiated, refed cultures. Also, the specific activity in vitro of DNA polymerase from irradiated refed cells is very much greater than that of polymerase from unirradiated, refed cells.

The degree of unwinding of the DNA helix by ethidium: I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients

A series of covalently closed bacteriophage PM2 DNA samples with varying degrees of superhelicity were prepared in vitro. The amount of bound ethidium per DNA nueleotide needed for the removal of all superhelical turns, vc0, was determined for each sample by a number of methods. In order to evaluate the unwinding angle for the binding of one ethidium molecule to a DNA double helix, the pH dependence of the buoyant densities in CsCI of these samples was examined. A new calibration relating the change in buoyant density of a DNA to the fraction of bases titrated has been obtained, by measuring the buoyant densities of a number of catenanes (interlocked rings) containing both single-stranded and double-stranded λ DNA rings, at a pH such that the single-stranded DNA is fully titrated while the double-stranded DNA is not titrated. This calibration was used to obtain the pH dependence of the fraction of DNA bases titrated for the phage PM2 DNAs with differing extents of supercoiling. A simple theoretical analysis shows that in a restricted pH range close to pHm, the melting pH of the DNA in the absence of the topological constraint associated with covalently closed double-stranded DNAs, the difference in the fraction of bases titrated at a certain pH between two covalently closed DNAs with different degrees of superhelicity is directly proportional to the difference in the vc0 values of the DNAs. The unwinding angle per bound ethidium molecule can be obtained from the proportionality constant. In this way, it is not necessary to know precisely the actual pH value for either DNA, pHe, at which the DNA is titrated to the extent that it contains no superhelical turns. The conclusion of the theoretical analysis and the experimental results is that the binding of an ethidium molecule to a double-stranded DNA unwinds the DNA helix by an angle φe = 26 °. The uncertainty in this value is estimated to be less than 10%. The new value for φe is approximately a factor of two larger than the value 12 °, which has been in use in the past decade. In the earlier alkaline titration results for polyoma DNA (Vinograd et al., 1968), which had been interpreted as supporting the 12 ° value, the calculation of φe was critically dependent on knowing pHe. It is believed that pHe was underestimated in the earlier work, resulting in a low φe value. Since the previous value φe = 12 ° has been widely used in the determination of the number of superhelical turns for many DNAs, and in measurements on the angular alterations of the DNA helix by the binding of a variety of small and large molecules and by solvent and temperature changes, the new value φe = 26 ° requires proportional adjustments of many previous results.

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.

Membrane synthesis in Bacillus subtilis. 3. The morphological localization of the sites of membrane synthesis.

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.

The effect of ethidium bromide on mitochondrial DNA synthesis and mitochondrial DNA structure in HeLa cells.

The synthesis of mitochondrial DNA (mDNA) in HeLa cells is selectively inhibited by relatively low concentrations of ethidium bromide. After exposure of cells to strongly inhibitory concentrations of the drug, the apparent superhelix density of mDNA is rapidly increased, as judged by its buoyant density in CsCl in the presence of ethidium bromide. Mitochondrial DNA synthesized in the presence of partially inhibitory concentrations of ethidium bromide is also altered in its buoyant density in the presence of the dye, but is more heterogeneous in this respect. However, the change in buoyant density of newly synthesized mDNA may be explained by changes in structure other than a change in superhelix density, as indicated by its increased resistance to digestion by pancreatic DNase.

Isopycnometric serology: a new technique based on buoyant density changes in latex beads.

WE wish to describe new serological techniques based on changes in buoyant density occurring when antibodies, or antigens, or both, are attached to latex beads. For the density change to be observed, the mass of the attached particles must be an appreciable fraction of the total mass. Using very small carrier beads less than one hundred antibody molecules or one virus particle per bead can be detected. The speed and sensitivity of the method make it directly applicable to the clinically important problem of rapid identification of infectious agents.

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.

The separation of different cell classes from lymphoid organs. 3. Te purfication of erythroid cells by pH-induced density changes.

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90-97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.

Early events in the infection process of adenovirus type 5 in HeLa cells

The fate of adenovirus type 5r after infection of HeLa cells has been studied with the help of purified labeled virus preparations. The infection process starts with the adsorption of virions on the cell surface followed by penetration into the cells. Shortly after penetration, the particles inside the cells have a similar appearance as intact virions. However, a small amount of protein must have been lost, as concluded from radioactivity measurements and from the change in buoyant density (1.35 g/cm 3 instead of 1.34 g/cm 3, the value for intact virions). Evidence is presented that antigen B is absent from the 1.35-particles. After the transformation of virions into 1.35-particles, further uncoating leads to the formation of DNase-sensitive DNA-protein complexes. The proteins released during the uncoating show a buoyant density of 1.31 g/cm 3 and are almost completely insoluble in acid. No extensive breakdown of protein can be detected. The processes of attachment, penetration, and uncoating are not inhibited by prevention of de novo protein synthesis, as could be shown with cycloheximide.

Alkylated DNA: Buoyant density changes and modes of decomposition

1. 1. Alkylation of DNA with nitrogen mustard ( N-methyl-bis(2-chloroethyl)-amine·HCl) causes a reduction in buoyant density. The magnitude of the effect depends on the amount of 14C-labeled nitrogen mustard bound and is independent of the DNA base composition. 2. 2. The relative hypodensities in CsCl, CsCl-LiCl (2:1 mole ratio), and Cs 2SO 4 were measured. A method for the analysis of this type of data is proposed. It was concluded that the buoyant density reduction is mainly due to a large increase in hydration, the extent of which was estimated. 3. 3. The increased hydration may be due to the presence of an ammonium ion on the bound nitrogen mustard moiety. The effect of pH on the hypodensity of nitrogen mustard reacted DNA supported this supposition. 4. 4. When nitrogen mustard reacted DNA is heated, the hypodensity is gradually lost concomitantly with a loss of bound 14C-labeled nitrogen mustard. In addition, the DNA breaks down to lower molecular weight fragments. The two modes of decomposition have different pH-dependences. The results, nevertheless, did not exclude a hypothetical two step decomposition process involving elimination of alkylguanine followed by strand scission.

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.