Alkaline Sucrose Density Gradient Research Articles

Identification of the short dispersed repetitive DNA sequences isolated from the zones of initiation of DNA synthesis in human cells as Alu-elements

DNA of Xeroderma pigmentosum cells was crosslinked in vivo with trioxsalen and long wave length ultraviolet light and the cells were cultured in the presence of labelled thymidine for one hour. The nascent DNA chains synthesized during this period and containing the DNA replication origins were isolated from the high molecular weight chromosomal DNA by an alkaline sucrose density gradient centrigugation. They were 5–10-fold enriched in short dispersed repetitive sequences identified by dot-blot hybridization to BLUR 8 plasmid as members of the human Alu-family.

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.

Mechanisms affecting peplomycin sensitivity of Chinese hamster cell lines.

Chinese hamster lung cell line V79 was ca. 13 times more resistant to peplomycin (PEP), and 6 times more resistant to bleomycin (BLM)-A2 than Chinese hamster ovary (CHO) cell line. The natural resistance of V79 cells to PEP or BLM was attributed to higher levels of BLM hydrolase activity and lower cellular uptake of the antibiotic. The sensitivity to PEP of a mutant clone CHO/O-2 T-1 was similar to that of CHO. A hybrid clone of CHO/O-2 T-1 X V79 showed an intermediate sensitivity to PEP between those of both parental cell lines, suggesting that the gene responsible for the natural resistance to PEP appears codominantly in the hybrid. The BLM hydrolase activity of the hybrid was also found intermediate between those of both parental cells. Mutant clones CHO/O-2 T-5 and CHO/O-2 T-6 were 8.3-9.0 times more sensitive to PEP than CHO cells. Hybrid clones CHO/O-2 T-5 X V79 and CHO/O-2 T-6 X V79 displayed PEP sensitivity similar to that of V79, suggesting that the gene responsible for the PEP supersensitivity (PEPss) behaves recessively in the hybrids. Both PEPss clones showed levels of BLM hydrolase and cellular uptake of [3H]PEP similar to the parental CHO cells, suggesting that the PEPss is due to neither BLM hydrolase nor cellular uptake of the antibiotic. Increased PEP-induced DNA cleavage and decreased DNA repair in the PEPss clones were demonstrated by alkaline sucrose density gradient sedimentation method. The results suggest that the PEPss of these mutant clones is attributed to decreased DNA-repairing activity and/or increased DNA-breaking activity.

The interplay between mixed function oxygenases and DNA alteration under PAH pollution

Our experience in DNA alteration assessment tells us that aquatic vertebrates, in general, and also some of the invertebrates, by living in polluted areas acquire altered DNA; DNA alterations being defined either as single-strand breaks, and/or alkali sensitive or single-strand specific nuclease-cleavable sites (sse). This can be shown by several methods: (i) Alkaline filter elution; 1 (ii) fluorescence analysis of DNA unwinding; 2 (iii) alkaline sucrose density gradient centrifugation; 3 (iv) alkaline unwinding analysis with hydroxyapatite differentiation; 4 (v) nucleoid sedimentation analysis; 5 (vi) supercoiled DNA relaxation detection by electrophoresis; 6 and (vii) measurement of DNA molecular weight distributions with the electron microscope. 7 The amount of sse per DNA mass can be a function of either an increased activity of DNA damaging agents or agents that inhibit DNA repair. Among DNA damaging agents are polycyclic aromatic hydrocarbons (PAH), that can cause sse by the ‘fast effect’ 8 or after induction upon uptake, through the action of mixed function oxygenase (MFO). Since MFO induction within certain limits is a function of PAH concentration measurement of this enzyme activity enables at least partial interpretation of the DNA alteration status, which can be a two-phase process.

Isolation and characterization of outer and cytoplasmic membranes from spheroplasts of Acetobacter aceti.

The spheroplast membrane of Acetobacter aceti IFO 3284 was separated into outer and cytoplasmic membranes by alkaline sucrose density gradient centrifugation after treatment of the cells with lysozyme in sucrose-EDTA, pH 8.0. The cytoplasmic membrane, which was transparent and red colored, showed a specific gravity of 1.15 g/ml, and a number of protein components. High contents of heme b and heme c, and high enzyme activities of various membrane-bound primary dehydrogenases, which are characteristics of acetic acid bacteria, were found in the cytoplasmic membrane fraction. On the other hand, the outer membrane, which was white and turbid when homogenized, exhibited a high content of 2-keto-3-deoxyoctonate, and only four major polypeptides were observed on SDS-polyacrylamide gel electrophoresis. The outer membrane showed a specific gravity of 1.25 g/ml due to its high lipopolysaccharide content. A predominant species of the outer membrane proteins, tentatively designated as AI, was found to be heat-modifia...

Autoradiographic studies of DNA replication in Werner's syndrome cells.

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werner's syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werner's syndrome cells, suggesting that there are two cell-cycle abnormalities in Werner's syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werner's syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werner's syndrome cells.

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: Comparison with the alkaline sucrose gradient centrifugation technique

An in vivo-in vitro alkaline DNA unwinding assay for single-strand breaks and/or alkalilabile lesions in hepatic DNA is described. The assay involves isolation of hepatic nuclei from mice, alkaline denaturation and unwinding of hepatic DNA, separation of single- and double-stranded DNA by hydroxylapatite batch chromatography, and quantitation of DNA in chromatographic fractions by a fluorometric assay. The method allows for the sensitive detection of hepatic DNA damage following in vivo administration of xenobiotics. Using this procedure, DNA fragmentation was demonstrated in alkali after administration of as little as 0.5 mg/kg of N-nitrosodimethylamine (DMN) to male B6C3F1 mice. A comparison of this technique with a similar alkaline sucrose density gradient centrifugation assay demonstrated comparable limits of sensitivity for the two procedures.

Gamma radiation induced single strand breaks in DNA and their repair in chloroplasts of Euglena cells: Comparison with nuclei of the light-grown and dark-grown cells

Exposure of light-grown cells of Euglena gracilis (Z strain) and chloroplasts in suspension to gamma radiation causes single strand breaks in the DNA of chloroplasts as assessed by sedimentation analysis in alkaline sucrose density gradients. Post-irradiation incubation of light-grown cells and chloroplasts at 25°C for 30 min results in restitution of the strand breaks in the DNA. Addition of deoxyribonucleoside triphosphates and ATP to the incubation system of the organelle does not have any effect on the efficiency of the DNA repair indicating the presence of sufficient reserves of the nucleotides required for repair functions in chloroplasts. The results of these in vitro experiments suggest that repair of the DNA in chloroplasts is brought about by independent machinery located in the organelle itself. Although the number of single strand breaks in DNA of chloroplasts is found to be less than that in the nuclei of light-grown or dark-grown cells, the difference concerning nuclei from the latter cells (cells devoid of chloroplasts) is 2-fold higher than that of nuclei from the former cells (cells with chloroplasts). The ability of chloroplasts to repair the damage in DNA is slightly better than the nuclei from light-grown cells, but it is significantly higher in comparison with that from nuclei of dark-grown cells.

Sensitization of Escherichia coli B/r to X-irradiation by 2'-chloro-2'-deoxythymidine.

The sensitizing effect of 2'Cl-TdR has been investigated from the viewpoint of the formation of free radicals at the C-2' position of 2'Cl-TdR when reacted with hydrated electrons. E. coli B/r cells were incubated in growth medium containing 2'Cl-TdR. Centrifugation experiments using CsCl equilibrium density gradients were carried out to confirm the incorporation of 2'Cl-TdR into DNA of the cells. The sedimentation profiles of DNA from cells grown in a medium containing 2'Cl-TdR were found at relatively heavier density positions in comparison with those of DNA from control cells. This result confirms that 2'Cl-TdR was incorporated into DNA. E. coli B/r cells which incorporated 2'Cl-TdR were more sensitive than the control cells for killing by X-irradiation under aerobic conditions. On the other hand, no difference in U.V.-inactivation curves between the cells grown in growth medium containing 2'Cl-TdR and the control cells was observed. From experiments using alkaline sucrose density gradient centrifugation, an increase in the frequency of radiation-induced single strand breaks of E. coli DNA containing 2'Cl-TdR was observed, compared with those of DNA from control cells. These results reveal that the sensitization of 2'Cl-TdR originates from an increase of damage at the sugar moiety in DNA.

Adenovirus DNA synthesized in the presence of aphidicolin.

Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals.

DNA breakage by methyl methanesulfonate and its repair in brain and liver cells cultured from fetal rat and mouse

DNA strand breakage and repair following methyl methanesulfonate (MMS) treatment of primary cell cultures from 14-day fetal Sprague-Dawley rat brain and liver and 12-day fetal C57BL/6 mouse brain and liver, were studied using alkaline sucrose density gradient analysis. Cells were incubated with MMS (7 mM or 14 mM) for 20 min and harvested for alkaline sucrose gradients 40 min or 24 h later. The extent of initial damage in fetal rat and fetal mouse cells was comparable. Fetal mouse brain and liver and rat liver showed nearly complete repair 24 h after treatment. However, fetal rat brain cells showed comparatively little repair after 24 h. The possible significance of a repair deficit in cultured rat fetal brain cells and the striking neurogenic organotropism of transplacentally administered direct-acting alkylating agents in the rat is discussed.

Effect of heparin on the porcine lymphocyte chromatin—II. comparative study of sedimentation of chromatin dna and isolated dna

1. 1. Sedimentation of chromatin DNA and isolated deproteinized DNA was compared in neutral and alkaline sucrose density gradients after incubation of chromatin or DNA with various concentrations of heparin. 2. 2. Irrespective of the molecular weight of DNA, an increase in the sedimentation constant of DNA was found with increasing concentration of the polyanion employed.

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells.

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.

Reevaluation of DNA chain elongation rate in human diploid fibroblasts

The rate of DNA chain elongation in human diploid fibroblasts (IMR90) of different ages was examined by DNA fiber autoradiography and alkaline sucrose density gradient centrifugation. There was no difference in chain elongation rate in various population doubling level cells.

Newly synthesised DNA in ageing human cells in culture treated with 4-nitroquinoline-1-oxide

Two lines of normal human embryonic lung fibroblasts, MRC-5 and F2002, were serially subcultured until senescence was attained. When cells were exposed to varying concentrations of the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO), the rate of DNA synthesis (as measured by thymidine incorporation) was reduced in a dosedependent fashion in cells from both early and late passages. While the overall amount of incorporation was considerably lower in old cells, the extent of inhibition caused by 4-NQO treatment (relative to appropriate controls) was not related to culture age. Alkaline sucrose density gradient analysis of newly synthesised DNA from cells pre-treated with 4-NQO failed to detect any significant variation in the size of labelled DNA from cells examined immediately after incubation with radioactive thymidine. The shift of this labelled material to high molecular weight in 4-NQO-treated cells also showed no age-related difference.

Isolation and characterization of pyocin R1 fibers.

By a mild alkaline treatment, pyocin R1 was disassembled into its structural parts, a contracted sheath (and its fragments), a core, and fibers. An alkaline sucrose density gradient centrifugation after this treatment was effective in obtaining fiber-density fractions. The pooled fractions were treated with IgGs against isolated sheaths and isolated cores, simultaneously, and then chromatographed on DEAE-Sepharose CL-6B. The final preparation of fibers purified in this way was confirmed to be homogeneous by electron microscopic observation and an immuno-precipitation reaction. The isolated fiber was found to consist of two major subunit proteins, No. 2 and No. 9, with molecular weights of 71,000 and 31,000, respectively. The fiber exhibited the ability to be adsorbed on sensitive bacterial cells (pseudomonas aeruginosa P14), and to protect against the inactivation of pyocin R1 by a lipopolysaccharide preparation from the bacteria.

Age-related variations in human lymphocyte DNA

The molecular weigth of DNA from human peripheral lymphocytes has been measured on alkaline sucrose density gradients. The number average molecular weight ( M N ) of DNA is found to decrease as the age of the donor increases. This result is discussed with reference to lesions in DNA, both repairable and non-repairable, which may accumulate with age.

Bleomycin induced inhibition of dna synthesis in ataxia-telangiectasia cell lines

Summary Exposure of fibroblast and lymphoblastoid cell lines from normal subjects to bleomycin (30–300 μg/ml) results in a dose dependent biphasic inhibition of DNA synthesis when compared with untreated cells. Using alkaline sucrose density gradient sedimentation we have confirmed that bleomycin decreases both DNA replicon initiation and chain elongation in normal lymphoblastoid cells. Doses of up to 100 μ/ml which caused a marked decrease in the rate of DNA synthesis in normal lymphoblastoid cells had much less effect on the inhibition of DNA synthesis in lymphoblastoid cell lines from ataxia-telangiectasia patients. The inhibition of DNA replicon initiation and chain elongation in these cell lines occurred only at high doses of bleomycin (100 μg/ml).

Isolation and characterization of virus-specific double-stranded DNA from tissues infected by bean golden mosaic virus

A double-stranded (ds) DNA which may be a replication intermediate was isolated from bean (Phaseolus vulgaris L. "Top Crop") leaves systemically infected with bean golden mosaic virus, a whitefly-transmitted plant virus with a genome of circular single-stranded (ss) DNA. The isolation method used phenol/chloroform extraction, hydroxyapatite column chromatography, and rate-zonal centrifugation. The dsDNA had sequences complementary to those of viral DNA. The guanine-plus-cytosine content was 35%, and the sedimentation coefficient in alkaline sucrose density gradients was similar to that of viral ssDNA. Digestion of the dsDNA by Hha I endonuclease produced fragments that corresponded exactly in number and size with those produced by complete digestion of circular viral ssDNA by Hha I, when the fragments were denatured and analyzed on polyacrylamide gels. The dsDNA molecule was a circular structure with one discontinuity in one strand; hybridization results suggest that some of a the dsDNA has a discontinuity in the viral strand and some has a discontinuity in the nonviral strand. On the basis of these structures for the dsDNA, a preliminary model for replication of viral DNA is discussed.

Lymphocyte DNA in Aplastic Anaemia

Using ultracentrifugation on alkaline sucrose density gradients the DNA of lymphocytes from 14 patients with aplastic anaemia and 23 controls was studied before and after exposure to bleomycin, an agent known to cause strand breaks in DNA. Before exposure to bleomycin the DNA from aplastic patients had more strand breaks than the DNA from the controls of similar ages. Following exposure to bleomycin an abnormal number of DNA strand breaks was produced in 10 of 14 patients and this molecular evidence of drug sensitivity correlated well with the sensitivity of the proliferating lymphocytes to bleomycin in tissue culture. Furthermore, two relatives of patients with aplastic anaemia showed similar abnormality of DNA before and after exposure to bleomycin and increased sensitivity to bleomycin in tissue culture. These results suggest that an abnormality of DNA structure and/or repair may be present in some patients with aplastic anaemia.