CsCl-ethidium Bromide Gradients Research Articles

Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients

Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients

Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients

Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients

Triggering of the Human Interleukin-6 Gene by Interferon-γ and Tumor Necrosis Factor-α in Monocytic Cells Involves Cooperation between Interferon Regulatory Factor-1, NFκB, and Sp1 Transcription Factors

We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.

Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA(1), hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA(2) and satellite DNA(3), recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.

Circular forms of viral DNA in Marek's disease virus-transformed lymphoblastoid cells.

The state of viral DNA in three Marek's disease virus (MDV)-transformed chicken cell lines (MDCC-MSB-1, MDCC-HP-2, MDCC-RP-1) was investigated by CsCl-density gradient centrifugation, and sedimentation analysis in neutral glycerol gradients. Each cell line contained MDV DNA integrated into the host cell genome. Additionally, free viral DNA could be identified in MDCC-MSB-1 and MDCC-HP-2 cells, sedimenting at about 100 S, and banding at the position of circular DNA in CsCl-ethidiumbromide gradients. Thus, MDV DNA with properties of circular plasmid DNA could be demonstrated in 2 virus-transformed cell lines. The significance of circular plasmid forms of viral DNA remains yet to be clarified, since these are apparently not regularly found in MDV-transformed cells.

Bacteriophage-induced Sporulation in Bacillus cereus T

An oligosporogenic strain of Bacillus cereus T was found to carry an inducible phage. CsCl-ethidium bromide gradients of lysates of this strain contained satellite DNA of the same density as the phage genome. Neither the phage nor the satellite DNA could be detected in the sporogenic parental strain. Purified phage preparations increased sporulation from 10-50-fold when added to cultures of the oligosporogenic strain.

Lytic cycle replicative forms of bacteriophages P1 and P1 dl: Concatemer forms

The permutedness of bacteriophage P1 chromosomes has implied that concatemers must be lytic cycle chromosomal forms (LCF). We have partially characterized LCF of P1 and a P1 dl, employing isopycnic fractionation in CsCl-ethidium bromide gradients and alkaline sucrose gradient centrifugation. The genetic identity of LCF was established by DNA filter annealings of radioactive LCF. In alkaline sucrose gradients, concatemer strands of LCF between 2 and 10 genome units long were recognized as DNA sedimenting slower than monomer supercoils and faster than single-strand monomers. Fractionation of pulse-labeled LCF showed that by 8 min after induction, most or all of the DNA synthesis proceeds by extension of concatemers. Pulse-chase experiments showed that the concatemer strands are part of persisting LCF. The P1 dl plasmid of M. E. Rae and M. Stodolsky ( Virology, 58, 32–54, 1974 ) was found to contain 133kb, as contrasted with 90 kb for the P1 plasmid. Concatemer strands were generated in thermally induced P1 dl lysogens, and had a higher average sedimentation rate than those of P1. In the P1-infected P1 dl lysogen, the sedimentation patterns of P1 and P1 dl strands were distinct. This fact indicated that recombination between P1 and P1 dl was not the major contributor to the formation of concatemer strands.

Partial characterization of viral DNA from human genital warts (Condylomata acuminata).

By centrifuging total cellular DNA derived from human genital warts (condylomata acuminata) in CsCl-ethidium bromide gradients, supercoiled DNA was isolated. The molecular weight of this DNA was determined by agarose gel electrophoresis and amounted to 5.1 X 10(4). This DNA isolated from an individual genital wart was annealed to fractions of aqueous supernatants of the same wart after prior centrifugation of this material in CsCl density gradients. Annealing was observed at a density of approximately 1.32 g/ml corresponding to the expected density of papilloma virus particles. Since such particles were also observed in the same preparation by electron microscopy, it was concluded that the supercoiled DNA molecules were derived from papilloma virus nucleocapsids. Positive hybridization was found with six additional preparations from individual genital warts. Therefore, it seems that the isolated DNA prevails in condylomata acuminata. The DNA is different from the other five types of human papilloma viruses described thus far in regard to its restriction endonuclease cleavage patterns. The virus analyzed is tentatively designated as human papilloma virus type 6 (HPV 6).

Covalently closed circular DNA molecules of low superhelix density as intermediate forms in plasmid replication.

COVALENTLY closed circular (CCC) duplex extrachromosomal DNAs are widespread in biological systems1. Because of their physical properties and small size, extra chromosomal circular DNAs are useful for studying the biology of genetic material. Their mechanism of replication is of particular interest and has been investigated extensively. Recent studies have shown that the replication of different types of circular DNA, such as plasmids of Escherichia coli, the DNA of polyoma and SV40 viruses, and mitochondrial DNA is basically similar2,3. Non-replicating circular DNA is isolated from cells largely as covalently closed supercoiled molecules (form I DNA). Because of its topological constraints, CCC DNA has a restricted affinity for intercalating dyes such as ethidium bromide and propidium diiodide; its buoyant density in gradients of CsCl containing such dyes is consequently much greater than that of non-circular or nicked circular (form II) DNA species, which can unwind to accommodate maximal amounts of dye4. DNA molecules in the process of replication have been shown to have buoyant densities intermediate to form I and form II DNA (refs 5–12 and L. Katz, P. H. Williams, S. Sato, R. W. Leavitt, and D. R. Helinski, unpublished). In a previous study of replicative forms of a constructed recombinant plasmid (pSC134) of E. coli13, we observed material pulse labelled with radioactive thymidine that had a buoyant density in CsCl–ethidium bromide gradients even greater than that of form I DNA5. Here we report results which suggest that this material, termed “heavy replicative DNA” or hrDNA, is a covalently closed circular DNA species that has a reduced number of supercoils, and is an intermediate in the replicative process.

Covalently closed circular DNA molecules deficient in superhelical density as intermediates in plasmid life cycle.

WE have described1 the replication of the conjugative R plasmid, RSF1040, a deletion mutant of R6K (ref. 2). RSF1040 possesses two distinct origins and replication generally proceeds from one or the other in an asymmetrically bidirectional mode to a unique terminus1, although in several instances molecules with two simultaneous initiations were observed2. During the course of examining the kinetic behaviour of replicating RSF1040 DNA (ref. 1), we observed 3H-thymidine pulse-labelled material that had a greater buoyant density in CsCl–ethidium bromide gradients than did mature covalently closed circular (form I) plasmid DNA. The nature of this material (‘heavy DNA’) and its relationship to the replication cycle are the subjects of this report.

Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.

Size, conformation and purity of chloroplast DNA of some higher plants

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g · cm −3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g · cm −3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 ± 2.1 μm for Antirrhinum, 45.7 ± 1.9 μm for Spinacia, 44.9 ± 1.7 μm for Beta and 45.2 μm for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

The genomic complexity of Acanthamoeba castellanii mitochondrial DNA.

DNA of DNase‐treated mitochondria of Acanthamoeba castellanii bands at a buoyant density of 1.690 g/cm3. Up to 80% of the molecules were circular, with a circumference of 12.7 μm. Two circular conformations, the closed twisted and the open form, were separated in preparative CsCl–ethidium bromide gradients. When mtDNA was renatured at a temperature Tm– 23°C, deviations from second‐order kinetics were observed. At Tm– 10°C, however, it renatured as a single kinetic class without indications of sequence repetition. The coincidence of the kinetic complexity (2.57 × 107) and the circle contour length within the limits of error of the methods suggests that each circular molecule represents a chondriome. The main DNA component, which is of nuclear origin and has a peak density of 1.716 g/cm3 in CsCl, displays heterophasic renaturation kinetics. Its slow‐reassociating fraction shows a kinetic complexity corresponding to a molecular weight of 2.1 × 1010. Approximately 22% of the main component appears to be repetitive at a renaturation temperature of Tm– 23°C.

State of the DNA-synthesizing system in isolated mitochondria from the mature eggs of the loach ( Misgurnus fossilis)

1. Isolated mitochondria from unfertilized loach eggs incubated in the reaction mixture for DNA synthesis with deoxynucleoside triphosphates incorporate 0.03 pmole of [ 3H]dTTP into the acid-insoluble fraction per mg of mitochondrial protein per h. This incorporation is at least 10 times lower than for mitochondria from later developmental stages of the loach when the biosynthesis of mitochondrial DNA is switched on. 2. Sedimentation analysis of the labeled incorporation product in CsCl-ethidiumbromide density gradients shows that essentially all radioactivity is present in the upper band of such gradients containing open DNA molecules. In contrast up to 80 % of radioactivity incorporated by mitochondria from later developmental stages of the loach is associated with lower and intermediate zones of the gradient containing molecules replicating on the covalently closed templates. 3. On the basis of 1 and 2 we conclude that loach egg mitochondria are inactive in replicative DNA synthesis and the small observed incorporation represents repair associated with spontaneous induction of single-strand breaks in mitochondrial DNA in the course of isolation and/or incubation of mitochondria. 4. Induction of single-strand breaks in mitochondrial DNA by treatment of isolated mitochondria with bruneomycin, known to produce among others 3′-OH termini in DNA, stimulates the incorporation of [ 3H]dTTP into acid-insoluble products 30–50 times. DNA labeled under these conditions is native, 90 % of the labeled molecules is present in the upper band of CsCl-ethidiumbromide gradients. The sedimentation coefficient of the labeled material in neutral sucrose is 27 S. 5. About 10 % of DNA labeled in the presence of a low concentration of bruneomycin (0.1 μg/ml) is associated with covalently closed molecules of mitochondrial DNA. Formation of this material is probably a consequence of sealing of labeled open circular DNA by a joining enzyme. This result may be evidence for the presence of DNA ligase in mitochondria from unfertilized eggs. 6. At least one explanation for the inactivity of egg mitochondria in replicative DNA synthesis is the absence of primer containing the 3′-OH terminal group necessary for the manifestation of DNA polymerase activity.

The resolution of polyoma DNA by polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is a valuable method for producing tine resolution of both single and double stranded RNA species [l-5] . In contrast to this, all DNA molecules larger than about lo6 daltons tend to move with the same electrophoretic mobility [l] , and no linear relationship between log molecular weight and electrophoretic mobility holds as well as it does for RNA [6,7] . On the other hand, some degree of success has been achieved in separating small DNA fragments [8,9] . Since the DNA of Polyoma virus is a double-stranded circular molecule, we have attempted to determine whether the difference in form between circular and linear DNA would provide a basis for separation by polyacrylamide gel electrophoresis. Two forms of the circular molecule can exist: the naturally-occurring supercoiled form, and one with a more relaxed open circular configuration resulting from one or more single-strand nicks [lo] (referred to hereafter as nicked circular DNA). At present, the most effective method for separating these is by isopycnic banding in CsCl-ethidium bromide gradients [ 1 l] . This method is time-consuming, but will separate supercoiled DNA from nicked circular and from linear forms, the latter two banding together at a lower density (and hence not themselves being separated by this method). We have been able to show that supercoiled and nicked