Theoretical analysis of gel electrophoretic data for interaction of lysine rich histone with supercoiled DNA.

We demonstrate the possibility of using gel electrophoresis as a technique for the quantitative analysis of interaction of lysine rich histone with DNA. On the basis of theoretical framework for extended ligand binding to one-dimensional lattices such as DNA we have set up systems of equations which relate the ligand-to-DNA ratio to the observed gel migration distance of the complex. From the analysis of experimental data for gel electrophoresis of supercoiled DNA in the presence of lysine rich histones we have found that the observed variation of electrophoretic mobilities of the histone-DNA complexes at low histone-to-DNA ratios can be described by a non-cooperative binding behaviour. At this limit we have estimated the intrinsic binding constant to be of the order of 10(3) M-1.     

Improved electrophoretic separation of supercoiled and relaxed DNA in the presence of ethidium bromide.

Supercoiled and relaxed DNA were resolved electrophoretically in the presence of 0.5 micrograms/ml ethidium bromide. Under these conditions the Gaussian distributions of topological isomers of both supercoiled and relaxed DNA migrated as discrete bands. The separation of these DNAs was optimized by varying the concentration of electrode buffer. Electrophoresis in the presence of 160 mM Tris-acetate, pH 8.3, 4 mM EDTA resulted in a 20-fold increase in the separation of relaxed and supercoiled DNA relative to electrophoresis in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA.     

Agarose gel electrophoretic evidence for domains of nuclear DNA linked with bonds cleavable with sulfhydryl molecules

Complexes of intact nuclear DNA with proteins undissociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage lambda DNA) into smaller-Mr forms. Following limited restriction endonuclease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60-110 kbp range. These 60-110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.     

碳酸钙沉淀法回收琼脂糖凝胶中DNA的探讨

采用碳酸钙沉淀法回收琼脂糖凝胶中的DNA,达到分离纯化目的,回收后的DNA可用于重组、PCR等研究。首先将含有目的DNA的琼脂糖凝胶用Nal溶液融解,然后加入cacl2,和NaHCO3,生成CaCO3,沉淀,DNA与cac03形成复合物,通过离心分离出沉淀复合物,利用稀酸溶解沉淀,再用无水乙醇沉降,即可回收目标DNA。利用该方法回收了质粒、毛白杨和转基因羊基因组DNA,同收率为20％～50％,0D260／OD280,为1．7～19,最大回收了21kb片段,最小回收250bp片段,回收后的DNA样品进行了PCR扩增和限制性内切酶反应,PCR可以扩增出目的片段,同时限制性内切酶可以将回收后的DNA切开,表明DNA质量良好。利用碳酸钙沉淀法可以回收琼脂糖凝胶中的DNA,此法简单、易行,较为有效。     

The binding of actinomycin D and adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug:DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of Φχ174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA0 is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

Dye titrations of covalently closed supercoiled DNA analyzed by agarose gel electrophoresis.

We have developed a rapid electrophoretic technique for performing ethidium bromide dye titrations in cylindrical 0.7% agarose gels. The technique was used to analyze the extent of supercoiling in circular covalently closed SV40, Co1E1, and pSC101 DNA. We have estimated the superhelical densities of SV40, Co1E1, and pSC101 DNA to be ?0.050, ?0.078, and ?0.085 respectively. The results obtained for native SV40 DNA correlate well with previously published values for the superhelical density of this DNA when these values are corrected to reflect a 26° duplex unwinding angle for ethidium bromide. Ethidium bromide concentrations sufficient to partially relax a supercoiled DNA allow the DNA to be resolved into a series of discrete bands in agarose gels. The distribution of bands represents a natural heterogeneity in the superhelical densities of the DNA molecules in the population.     

Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium

We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Agarose gel electrophoresis and restriction endonuclease analysis of largeStaphylococcus plasmids

Preliminary studies using an improved method for the agarose gel electrophoresis of semipurified, cleared lysates of staphylococci have indicated some distinct differences in plasmid composition between the coagulase-positive speciesStaphylococcus aureus andStaphylococcus intermedius, and various coagulase-negative species. Penicillinase-positive strains ofS. intermedius andS. simulans did not carry large penicillinase plasmids like most penicillinase-positive strains ofS. aureus. Most coagulase-negative species examined demonstrated complex plasmid profiles. Codigestion by the restriction endonucleasesHaeIII andHpaI offered a useful approach for “fingerprinting” large plasmids from various strains and species.     

Temperature dependence of the gel electrophoretic mobility of superhelical DNA.

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a function of both temperature and of linking number (Lk). At temperatures below 37 degrees, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37 degrees the electrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37 degrees - 65 degrees the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37 degrees and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6 degrees, which lies within 3 degrees of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.     

Strand scission of DNA by bound adriamycin and daunorubicin in the presence of reducing agents.

Adriamycin and daunorubicin bound to covalently closed circular DNA nick the latter when reduced by sodium borohydride as demonstrated using an ethidium bromide fluorescence assay. The degradation, dependent on oxygen, is strongly inhibited by (i) superoxide dismutase (ii) catalase and (iii) sodium benzoate indicating the intermediacy in the cleavage of superoxide radical anion, hydrogen peroxide and hydroxyl radicals respectively. Less nicking of the DNA is observed by the reduced aglycones, so binding to the DNA by the aminosugar moiety assists the cleavage process. Adriamycin, daunorubicin and both ring C reduced forms bind intercalatively and completely relax supercoiled DNA. The results provide a possible rationale for the degradation of DNA which accompanies anthracycline administration.     

DNA damage induced by ascorbate in the presence of Cu2+

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.     

Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.

Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.     

Glucose 6-phosphate dehydrogenase inDrosophila melanogaster: Starch gel electrophoretic variation due to molecular instability

Two starch gel electrophoretic variants of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogasterwere partially purified and characterized biochemically. The difference in their migration on starch gel is not due to a charge difference, but rather to a difference in molecular size due to instability of one of the variants. Crosses between the two variants did not produce offspring showing an intermediate electrophoretic band in the heterozygous female. That an hybrid molecule does exist in the heterozygous female, however, was demonstrated by taking advantage of differences in heat lability and structural stability of the two variants. Hence, we conclude that the G6PD locus in both X-chromosomes is active in each cell in female Drosophila.Our findings with DrosophilaG6PD emphasize the importance to protein variation and enzyme function of mutations leading to amino acid substitutions which do not produce a change in net charge. Aided by National Institutes of Health grants HD 00486, HD 00004, and GM 14155.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.