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Biomedical subjects

E Palecek

Publications and source records attributed to E Palecek.

At least 37 records · Page 2Linked to original sources
PubMed1995

Complex of osmium tetroxide with 1,10-phenanthroline binds covalently to double-stranded DNA.

Complex of osmium tetroxide with 1,10-phenanthroline (Os,phen) reacts with double-stranded B-DNA in contrast to osmium tetroxide, pyridine and other osmium structural probes which show a strong preference for single-stranded DNA (ssDNA) (Palecek, E. in Abelson, J.N., and Simon, M.I. (eds), Lilley, D.M.J., and Dahlberg, J.E., (volume eds.), Methods in Enzymology, Vol. 212, DNA Structures, part B., Academic Press, 139-155 (1992)). Modification of negatively supercoiled DNA (scDNA) with Os,phen changes the DNA electrophoretic mobility inducing the DNA relaxation at lower degrees of modification followed by formation of positive supercoils at higher modification extents. Electrophoretic mobility of the Os,phen-modified DNA fragments in agarose gel is almost unchanged while a strong retardation of the same fragments is observed in polyacrylamide gels. Os,phen-modified DNA is hypersensitive to nuclease S1. Cleavage of this DNA by restriction enzymes is selectively inhibited showing a preference of Os,phen for TA and AT dinucleotide steps. DNA modification by Os,phen is inhibited by low and moderate concentrations of MgCl2. The covalent binding of Os,phen to double-stranded DNA (dsDNA) is preceded by noncovalent interactions (probably intercalation) inducing DNA structural changes; the shape of the Os,phen-modified DNA molecule appears to be severely deformed.

Base Composition↗
PubMed1993

Conformational junctions between left-handed DNA in (dA-dT)16 and contiguous B-DNA in a supercoiled plasmid contain chemically reactive bases.

Alternating adenine-thymine sequences in supercoiled DNA may undergo a transition to the left-handed Z-conformation in the presence of Ni2+ ions and high Na+ concentrations [(1989) FEBS Lett. 243, 313-317]. In this work we have studied the junctions between B- and Z-conformations in a supercoiled plasmid containing a (dA-dT)16 insert, by means of chemical probing. We observed enhanced reactivity of bases at both ends of the alternating tract to chloro- and bromoacetaldehyde. The degree of chemical reactivity was found to increase with the level of negative supercoiling. Only individual bases were observed to be reactive in the B-Z junctions, consistent with tightly localized interfacial regions.

Acetaldehyde↗
PubMed1993

Cyclic voltammetry of submicrogram quantities of supercoiled, linear and denatured DNAs with DNA-modified mercury electrode.

We have previously shown that a DNA-modified electrode can be prepared by immersing the hanging mercury drop electrode (HMDE) in a small volume (about 5-10 microliters) of a DNA solution. Within a short period of time the DNA is irreversibly adsorbed at the electrode, resisting subsequent washing. The electrode is then transferred into a voltammetric cell filled with the background electrolyte (that does not contain any nucleic acid) in which the voltammetric measurements are performed. This procedure is called adsorptive transfer stripping voltammetry (AdTSV). In this paper AdTS cyclic voltammetry (CV) peaks of DNA were measured to report on the stability of the attachment of plasmid DNA molecules to the electrode surface. It was shown that the attachment of plasmid DNA to the electrode was, like the case of calf thymus DNA, sufficiently stable. If the DNA-modified electrode was immersed in a protein solution no significant exchange between the DNA and protein was observed. Submicrogram amounts of DNA were sufficient to attain full coverage of the electrode at relatively short waiting times and the detection limit of the denatured DNA was below 2 ng. The intensity of the AdTS CV signals of supercoiled, linearized and thermally denatured linear DNAs differed from one another. It was shown that alkaline denaturation of linear and supercoiled DNA in solution can be studied using AdTS CV technique. It was further shown that at neutral pH the linearized plasmid undergoes denaturation due to a prolonged contact with the electrode charged to potentials around -1.2 V. Such a surface denaturation has been previously observed with calf thymus DNA and synthetic double-stranded polynucleotides. Our results show that in contrast to linear DNA, supercoiled DNA was not significantly denatured as a result of its prolonged contact with the electrode charged to various potentials in the range between -0.1 to -1.5 V.

Adsorption↗
PubMed1993

Local opening of the DNA double helix in eukaryotic cells detected by osmium probe and adduct-specific immunofluorescence.

The structure of DNA in mouse fibroblast 3T3 cells has been investigated with the single-strand-selective probe, the complex of osmium tetroxide and 2,2'-bipyridine (Os,bipy). DNA-Os,bipy adducts in the cells were detected by immunofluorescence using a highly specific, affinity-purified polyclonal antibody. Treatment of living cells with the chemical probe led to a distinct but nonuniform nuclear staining. We attribute the positive nuclear staining to the existence of single-stranded and distorted DNA regions in the living cell. Confocal laser scanning microscopy revealed dark areas corresponding to nucleoli and regions of condensed chromatin. These conclusions were supported by the results of experiments in which the chemical probe was applied to fixed cells treated with 45% acetic acid or with acidic buffers (pH 1.8-2.5). An inverse staining pattern was obtained, characterized by intense immunofluorescence of the condensed chromatin regions. Thus, the structural transitions and/or chemical alterations (e.g. depurination) induced by acid treatment increase the accessibility of normally unreactive DNA bases. We conclude that open DNA structures recognized by the chemical probe in the cells prior to their fixation are contained mainly in decondensed and transcriptionally active chromatin, but are virtually absent from nucleoli and condensed chromatin.

2,2'-Dipyridyl↗
PubMed1991

Probing of DNA structure with osmium tetroxide,2,2'-bipyridine. Adduct-specific antibodies.

Antibodies against DNA modified with a single-strand selective probe, OsO4 in complex with 2,2'-bipyridine (Os,bipy), were raised in rabbits. These antibodies were fractionated using affinity column chromatography and fractions S89-II and S89-III characterized as highly specific for DNA-Os,bipy adduct with no cross reactivity to at least 1000-fold excess of unmodified DNA, RNA and Os,bipy-modified and unmodified proteins. Cross-reactivity to Os,bipy-modified RNA was very small. S89-II showed no cross-reactivity to DNA modified with OsO4 complexed with tetramethylethylenediamine or with bathophenanthroline disulphonic acid and to DNA oxidized with KMnO4. It cross-reacted, however, with DNA modified with OsO4,1,10-phenanthroline complex. The limit of detection of immunodot-blot analysis of extensively Os,bipy-modified DNA was below 0.5 pg. Small extent of Os,bipy-modification of supercoiled and linearized plasmids can be detected by DNA gel retardation and immunoblotting techniques. E. coli cells contain DNA regions in which bases are accessible to the single-strand selective probe.

2,2'-Dipyridyl↗
PubMed1991

Local supercoil-stabilized DNA structures.

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.

Base Sequence↗
PubMed1991

Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.

Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.

2,2'-Dipyridyl↗
PubMed1990

Protonated triplex DNA in E. coli cells as detected by chemical probing.

The triplex structure in vitro is well established; however, no direct evidence has been available concerning its existence in the cell. Using the direct chemical probing here we show that the triplex H structure can exist in E. coli cells at acidic intracellular pH values; this structure differs in some details from that observed in vitro.

Base Sequence↗
PubMed1990

Superhelical torsion in cellular DNA responds directly to environmental and genetic factors.

Superhelical tension of DNA in living bacteria is believed to be partially constrained by interaction with proteins. Yet DNA topology is a significant factor in a number of genetic functions and is apparently affected by both genetic and environmental influences. We have employed a technique that allows us to estimate the level of unconstrained superhelical tension inside the cell. We study the formation of cruciform structures by alternating adenine-thymine sequences in plasmid DNA by in situ chemical probing. This structural transition is driven by superhelical torsion in the DNA and thus reports directly on the level of such tension in the cellular DNA. We observe that the effect of osmotic shock is an elevation of superhelical tension; quantitative comparison with changes in plasmid linking number indicates that the alteration in DNA topology is all unconstrained. We also show that the synthesis of defective topoisomerase leads to increased superhelical tension in plasmid DNA. These experiments demonstrate that the effect of environmental and genetic influences is felt directly at the level of torsional stress in the cellular DNA.

Base Composition↗
PubMed1989

Probing of DNA structure with osmium tetroxide. Effect of ligands.

Fourteen OsO4 complexes with different ligands were tested as probes of DNA structure. Of these complexes, only OsO4-2,2'-bipyridine (Os-bipy), OsO4-bathophenanthrolinedisulfonic acid (Os-bpds) and OsO4-N,N,N',N'-tetramethylenediamine (Os-TMEN) site-specifically modified the ColE1 cruciform in a supercoiled plasmid pColIR215 at millimolar concentrations. Os-bipy, Os-bpds and Os-TMEN also displayed site-specific modification of the B-Z junctions in the supercoiled plasmid pRW751 containing (dC-dG)n inserts.

Bacteriocin Plasmids↗
PubMed1989

Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+.

The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).

DNA Transposable Elements↗
PubMed1989

Electron microscopy of supercoiled pEJ4 DNA containing homopurine.homopyrimidine sequences.

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine.homopyrimidine tract assumes an unusual conformation) a single thick "stem" or a "denaturation bubble" in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The "denaturation bubbles" were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only "stems". The "stems" were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine.homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.

Base Sequence↗
PubMed1989

Antibodies to DNAs chemically modified with osmium structural probes.

It has previously been shown that osmium tetroxide, pyridine (Os,py) and osmium tetroxide, 2,2'-bipyridine (Os,bipy) are powerful probes of the DNA structure. To increase the possibilities of the detection of osmium-modified DNAs polyclonal antibodies against DNA modified with Os,py and Os,bipy were elicited in rabbits. Specificity of these sera or purified IgG was tested by ELISA and retardation of the DNA electrophoretic mobility in agarose gels. Antibodies against DNA-Os,py (anti-DNA-Os,py) reacted with single-stranded and double-stranded DNA-Os,py but they did not react with unmodified DNA; with DNA-Os,bipy only a weak reaction was observed. The specificity of the anti-DNA-Os,bipy was similar. Competition experiments with anti-DNA-Os,py showed a weak reaction with RNA-Os,py but no reaction with osmium-modified proteins and unmodified proteins and RNA. The results suggest that anti-DNA-Os,py may become an important tool in studies of DNA structure in situ.

Animals↗
PubMed1989

Probing of B-Z junctions in recombinant plasmids in vitro and in the cell with different osmium tetroxide complexes.

Complexes of OsO4 with 2,2'-bipyridine (Os,2,2'-bipy),4,4'-bipyridine (Os,4,4'-bipy), 1,10-phenanthroline (Os,phe), bathophenanthroline disulfonic acid (Os,bpds) and OsO4, pyridine reagent (Os,py) were used to probe structural distortions at the junctions between right-handed B and left-handed Z DNA in supercoiled plasmids pRW751 and pPK1 (both containing (dC-dG)13 and (dC-dG)16 segments). With all five complexes the site-specific modification at the B-Z junctions was detected in vitro but only Os,2,2'-bipy and Os,bpds produced strong site specific modification at submillimolar concentrations. In addition to the B-Z junctions. Os,phe also reacted at other sites. With the exception of Os,2,2'-bipy no one of the tested OsO4 complexes has proved to be suitable for probing structural distortions at the B-Z junctions in E. coli cells.

DNA, Bacterial↗
PubMed1988

Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution.

Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.

Animals↗