Neutral Sucrose Density Gradients Research Articles

DNA strand breaks induced by ionizing radiation on Rubrobacter radiotolerans, an extremely radioresistant bacterium

Rubrobacter radiotolerans is the most radioresistant bacterium showing 16kGy as D 37 against gamma-rays. However mechanisms of the radioresistance have been still unclear. To clarify the post-irradiating events in the cell, we investigated DNA strand breaks which is the most major lesion induced by ionizing radiation. The neutral sucrose density gradient centrifugation analysis showed that size of the chromosomal DNA gradually decreased with irradiation dose. However, the frequency of DNA strand breaks after ionizing irradiation was significantly lower than those reported for other eubacteria. The reduced DNA sizes were not recovered during the post-irradiating cultivation. These results suggest that in this organism DNA protection from damage is mainly involved in the radioresistance, but not DNA repair activities.

Changes in DNA supercoiling in fibroblasts cultured in the presence of hydralazine

We have analyzed changes in the supercoiling of nucleoid DNA of murine fibroblasts cultured in the presence of hydralazine. The entire DNA attached to the nuclear matrix was extracted from the cells and sedimented in neutral sucrose density gradients containing ethidium bromide. Nucleoids from cells treated with hydralazine responded to increasing ethidium bromide concentrations in a different way than those from control cultures. That is, supercoiled loops of DNA unwound with lower concentrations of ethidium bromide sedimented less rapidly than those of control cells, indicating that hydralazine reduced the degree of DNA supercoiling. Also, nucleoids from the drug-treated cells resisted the transition from relaxed to positive supercoiling at higher concentrations of ethidium bromide. Changes in nucleoid DNA supercoiling correlated directly with the dose of hydralazine in the fibroblast culture.

Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells ( A. P. Johnstone, and G. T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-γ-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.

DNA breakdown of 3T3 and SV 40-transformed 3T3 cells cultured in suspension

It was examined what effect of suspension culture exerted on prelabeled DNA of 3T3 and SV 40 transformed cells (SV3T3). On an alkaline sucrose density gradient the small size DNA of 3T3 cells increased with time of suspension, while that of SV3T3 did not. Furthermore, it was demonstrated that prelabeled DNA of suspended 3T3 cells became small on a neutral sucrose density gradient, in an alkaline and a neutral elution. When SV3T3 cells were treated with dimethylsulfoxide, the smaller DNA appeared on an alkaline sucrose density gradient.

A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts

A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage λ cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2–1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibriblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m 2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.

Effect of heparin on DNA

It was found that heparin is capable of increasing the sedimentation coefficient of DNA over alkaline and neutral sucrose density gradients as well as of reducing the intrinsic viscosity of native DNA. Irreversible compactization of DNA induced by heparin apparently by means of intramolecular aggregation of DNA with involvement of residual protein underlies the above-described phenomena. The influence of heparin on DNA is potentiated in more concentrated solutions. This should be taken into account in the assay of DNA-containing systems in the presence of heparin.

Fate of Unintegrated Viral DNA in Fv-1 Permissive and Resistant Mouse Cells Infected with Murine Leukemia Virus

We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1 resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1(b/b) (JLS-V9 and SIM.R) and Fv-1(n/n) (NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1 resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1 permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1 resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1 gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1 gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1 restriction does not operate through a degradation process. Therefore, the Fv-1 gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.

Microfluorometric filter determination of DNA in sucrose density gradients

A simple filter method for the fluorometric estimation of DNA in alkaline and neutral sucrose gradient fractions with DABA·2HCl is discussed. Alpha-450 membrane filters of regenerated cellulose (Gelman Instrument Co., Ann Arbor, Michigan) were used. In the proposed method washing of the DNA precipitate as well as the reaction with DABA·2HCl were performed directly on the filters, thus avoiding repeated washing and centrifugations of DNA precipitates applied hitherto in analogous fluorometric techniques. A good coincidence of the results concerning localization of DNA sedimentation profiles determined by radioisotopic and fluorometric methods was obtained. The method is very convenient for DNA estimation in alkaline and neutral sucrose density gradient fractions obtained by ultracentrifugation of DNA of nonproliferating cells where DNA labeling is very difficult, and in the case of human lymphocytes even impossible without stimulation for blastic transformation. Its other advantages are a considerably simplified procedure and a higher precision with respect to other fluorometric methods for determination of DNA.

Studies on the mechanism of action of ACNU, 1-(4-amino-2-methylpyrimidine-5-yl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride: effects on cultured HeLa S3 cells.

Treatment of cultured HeLa S3 cells with ACNU, 100 microgram/ml, for 30 min inhibited the cell growth intensely. The cell number was minimum on the 4th day after the treatment and recovered gradually, but it was still 24% of control of the 14th day. The colony formation, as an index of proliferation ability of cells, was remarkably inhibited and the number of colony formed on the 14th day after the treatment was 8% of control. DNA synthesis was inhibited by 59%, whereas little or no effect was observed on RNA or protein synthesis after 24 hr. Ethylene-14C-ACNU was bound to DNA and RNA to a similar degree, and that bound to the acid-insoluble fraction within 30 min was decreased by half after 24 hr and sustained thereafter. A significant decrease in sedimentation velocity of DNA on alkaline sucrose density gradient centrifugation was observed after 24 hr, and the decrease on neutral sucrose density gradient centrifugation was noticeable already by 2 hr. From the results of this study and other reports, the mechanism of action of ACNU seems to be alkalization of DNA followed by damage to DNA, which progresses quite slowly compared with other alkylating agents, causing cell damage and cell death.

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.

Infectivity of mengovirus replicative form. IV. Intracellular conversion into replicative intermediate

The replicative form (RF) of mengovirus was previously shown to be infectious, and the infectivity of this virus-induced, double-stranded RNA, to depend on the host-cell macromolecular synthesis. Analysis of the 3H-labeled RNAs recovered from the cytoplasm of 3H-RF-infected cells revealed that the parental RF molecules sedimented faster in neutral sucrose density gradients as the infectious cycle proceeded. When 3H-RF-infected cells were pulse-labeled with [ 14C]uridine at 3 hr after infection most of the [ 14C]RNA was found in a rapidly sedimenting structure derived from (or tightly associated with) the parental 3H-RF. This structure had all the characteristics of the replicative intermediate (RI). Treatment of the host cell with interferon abolished the infectivity of RF, but did not prevent the intracellular conversion of RIP into RI. By contrast, exposure of the cultures to actinomycin D suppressed both the infectivity of RF and its transition to RI. The role of a host cell factor in the mechanism of infectivity of picornavirus RF is discussed.

Identification and characterization of fast-sedimenting SV40 nucleoprotein complexes

Simian virus 40 (SV40) nucleoprotein complexes were extracted from nuclei of infected monkey cells and fractionated on neutral sucrose density gradients. Four forms of SV40 chromatin were distinguished by their labeling kinetics and apparent sedimentation coefficients: Replicating viral DNA in 95 S SV40 chromatin was converted to 75 S chromatin when viral DNA replication was completed. A portion of the 75 S chromatin was rapidly assembled into 200 S nucleoprotein complexes and subsequently into 240 S complexes. The 75 S and the fast-sedimenting complexes were further characterized and compared with mature SV40 virus by equilibrium density centrifugation, plaque assay, and electron microscopy. These experiments suggest that the fast-sedimenting forms may represent virion precursors or a subclass of salt-labile virions, but they contain little or no mature virus.

Evidence that incorporation of inorganic [ 32P]phosphate into genetic and/or metabolic DNA of regenerating rat liver is stimulated 6 hours earlier than similar incorporation of labeled thymidine

The onset of stimulated DNA synthesis in regenerating rat liver was studied by pulsing either [ Me- 3H]thymidine (dThd) or 32P i into rats at various intervals post-partial hepatectomy. Controls were rats subjected to laparotomy. After isolating DNA by 3 different methods, hydrolyzing the DNA enzymatically and chromatographically separating the constituent nucleotides, it was concluded that incorporation of 32P i into DNA was significantly stimulated 12 h post-partial hepatectomy, and that similar stimulation of [ Me- 3H]dThd incorporation occurred 6 h later, i.e., 18 h post-partial hepatectomy. The difference in the entry-time into DNA by the 2 precursors was not associated with mitochondrial DNA synthesis, perturbations of intracellular pools or unique juxtaposition of diurnal rhythms. Incorporation of 32P i into DNA 12 h post-partial hepatectomy was inhibited by 4 of 5 drugs known to inhibit, in other cell or tissue systems, the incorporation of labeled dThd into DNA and/or cell multiplication. Nuclei from 12 h regenerating rat liver incorporated [Me- 3 H]dThd-5′-P 3 into DNA in vitro at more than twice the rate determined for nuclei from 12-h laparotomized rats and after centrifugation through neutral sucrose density gradients, isotope from [Me- 3 H]dThd-5′-P 3 was distributed among all sedimentation species detectable by 260 nm absorbancy. Liver DNA was labeled 12 or 24 h post-partial hepatectomy with a 1 h pulse of 32P i or [ Me- 3H]dThd, respectively. After purification of the DNA and centrifugation through neutral sucrose density gradients, about 70% of 32P-labeled DNA was associated with fractions that absorbed light at 260 nm, while essentially all the 3H-labeled DNA was associated with such fractions. Both 32P-labeled DNA and 3H-labeled DNA were labile to DNAase hydrolysis. Liver DNA also was labeled with [ Me- 3H]dThd the first 20 days post-partum; 10 weeks later, these rats were subjected to partial hepatectomy; 12, 18 and 24 h post-operation, they were pulsed 1 h with 32P i. When purified DNA from these rats was centrifuged through neutral sucrose density gradients, profiles for 3H-radioactivity and absorbancy at 260 nm were superimposable, irrespective of time after operation. At 12, 18 and 24 h post-partial hepatectomy, 20, 80 and 90%, respectively, of 32P-radioactivity was associated with fractions that absorbed light at 260 nm and the specific radioactivity of this 32P-labeled DNA increased; the balance of the 32P-radioactivity was associated with fractions that sedimented more slowly than those showing 260 nm absorbancy. The distribution of 32P-radioactivity, incorporated 12 h post-partial hepatectomy, among pyrimidine isostichs was similar to the distribution of radioactivity from [ Me- 3H]dThd incorporated into DNA during the first 20 days post-partum. When hepatic cells from 12-h laparotomized or partially hepatectomized rats were lysed over and centrifuged through alkaline sucrose density gradients, about twice as much radioactivity was located in high molecular weight fractions from 12-h regenerating cells, and the high molecular weight DNA from either source was about equally labile to hydrolysis by DNAase or hot acid. It was concluded that the synthesis of genetic and/or metabolic DNA was stimulated 12 h post-partial hepatectomy and this stimulation occurred in the absence of demonstrable participation by exogenous [ Me- 3H]dThd.

The genomic RNA of Mengovirus II: Comparative analysis of the standard and denser virions

The standard Mengovirus particle (buoyant density, 1.34 g/ml) was separated from a denser one (1.44 g/ml) by isopycnic centrifugation in CsCl at pH 8.0. When sedimented through a neutral sucrose density gradient, the standard virions had an apparent sedimention coefficient of 190 S, whereas the denser particles were resolved into two unequal peaks at 220 and 190 S. The denser particles were infectious, and the proportion of denser to standard virions remained unmodified after 24 serial passages at a high m.o.i. The progeny of both the standard and denser particles contained the same relative proportion of standard and denser virions. 32P-labeled RNA extracted from denser particles was degraded to a variable extent. However, all of the big T 1 oligonucleotides characteristic of the standard Mengovirus genome were also present in the fingerprints of the RNA from the denser particles. These two findings suggested that the RNAs of the standard and denser particles were indeed the same, but that the general configuration of the denser virions made their RNA more accessible for interaction with the cesium salt and more susceptible to nuclease degradation. Analysis by SDS-PAGE of the 35S-labeled structural proteins revealed that the denser virions lacked a polypeptide with an apparent MW of 59,000. The remaining polypeptides were present, although their relative proportions were somehow altered. The significance of the denser particles is discussed.

DNA polymerase α is associated with replicating SV4O nucleoprotein complexes

Simian virus 40 (SV40) nucleoprotein complexes were extracted from nuclei of infected monkey cells and fractionated on neutral sucrose density gradients. Complexes which contained replicating SV40 DNA (95S) separated well from those containing closed circular supercoiled viral DNA (75S). DNA polymerase activity was associated with the replicating nucleoprotein complexes but not with the slower sedimenting complexes. This DNA polymerase activity coprecipitated with the nucleoprotein complexes in the presence of MgCl2 and remained associated with the 95S complexes. This DNA polymerase activity has been identified as primarily DNA polymerase alpha on the basis of its sedimentation behavior, optimum salt concentration, and sensitivity to N-ethylmaleimide. DNA polymerase gamma activity was also detected in the complexes, but DNA polymerase beta was not associated with the complexes.

Damage of DNA and its recovery in AH-109A cells treated with carboquone in vivo.

A single injection of carboquone at a dose of 0.1 mg/kg body weight induced damage of DNA of AH-109A cells as revealed by alkaline and neutral sucrose density gradient centrifugation. A significant decrease in the sedimentation velocity of DNA on alkaline sucrose density gradient centrifugation was observed 15 min after the injection, and it returned to that of control after 60 min. Thereafter, the size of DNA decreased progressively. When the cells were lysed with 2% sodium dodesyl sulfate solution and analyzed on neutral sucrose density gradient centrifugation, a similar change in the sedimentation velocity was observed. The results obtained from the studies of intraperitoneal growth of AH-109A cells pretreated with carboquone in vivo and the survival of host animals well corresponded to the extent of the damage of DNA as revealed by alkaline and neutral sucrose density gradient centrifugation.

Chromosome structure of Escherichia coli mutants temperature sensitive for deoxyribonucleic acid replication

Folded chromosomes were isolated from Eschericia coli thermosensitive dnaA initiation mutants incubated at the nonpermissive temperature and were analyzed by neutral sucrose density gradient centrifugation. A chromosomal structure that sedimented at approximately 1,500S accumulated when the dnaA gene product was inactive. When the cells were returned to a permissive temperature, the folded chromosomes exhibited a decrease in sedimentation velocity to 1,300S but still retained their uniform structure. Very little deoxyribonucleic acid synthesis occurred during the period in which the chromosomes exhibited the reduction in sedimentation velocity. A dnaG elongation mutant showed no unique chromosome structure when the dnaG gene product was inactive.

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10 7 daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10 8 daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10 6 daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.