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K Nejedlý

Publications and source records attributed to K Nejedlý.

At least 19 recordsLinked to original sources
PubMed2001

Genomic DNA regions whose complementary strands are prone to UV light-induced crosslinking.

We prepared an extensive set of DNA restriction fragments, irradiated them with UV light, and detected crosslinked complementary strands by electrophoresis in denaturing agarose gels. These experimental data were quantified by densitometry to determine tetranucleotide contributions to crosslinking. The tetranucleotide contributions were used to predict genomic maps of the crosslinking probability that permitted us to identify two strongly crosslinking genomic regions having 295 and 389 base pairs in length. The two sequences shared the (ATTTTATA).(TATAAAAT) octamer, which is a candidate for the hotspot of UV light-induced crosslinking between the complementary strands of DNA.

DNA↗
PubMed2001

Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA.

UV light crosslinks the complementary strands of DNA. The interstrand crosslinks may contribute to the biological and pathological effects that UV irradiation is known to bring about. Here alkaline agarose gel electrophoresis was used to assess the crosslinked fraction of 31 selected restriction fragments of six viral and plasmid DNA molecules exposed to UVC light irradiation. As many as 17 independent experiments were performed with the particular DNA fragments to get sufficiently precise data suitable for quantitative analyses. The data were used to determine how the crosslinked fraction depended on the dinucleotide, trinucleotide and tetranucleotide contents of the irradiated DNA fragments. This analysis demonstrated that DNA conformation and/or flexibility, rather than the local double helix thermostability, governed the phenomenon of crosslinking. For example, (GA).(TC) suppressed the crosslink formation in DNA more than any dinucleotide composed of only G and C. In addition, (CTAG).(CTAG) promoted crosslinking much more than any other tetranucleotide, including e.g. (TATA).(TATA), whereas the closely related (CATG).(CATG) belonged among the tetranucleotides that most suppressed the UV light induced crosslinks between the complementary strands of DNA. The present data reproduced crosslinking of the analyzed 31 restriction fragments with a correlation coefficient exceeding 0.90. This result will be useful to predict crosslinking along the whole human genome.

Base Sequence↗
PubMed1998

Analysis of a curved DNA constructed from alternating dAn:dTn-tracts in linear and supercoiled form by high resolution chemical probing.

Complex of osmium tetroxide and bipyridine (Os,bipy), KMnO4, and diethyl pyrocarbonate (DEPC) were used to probe curved DNA at single nucleotide resolution. The DNA was constructed from repeated dAn:dTn-blocks with dATATA and dAGAGA interblock sequences. The DNA was probed in the linear and supercoiled form at various salt concentrations. While all purines were available for DEPC attack, the thymines within the blocks were resistant to chemical probing by KMnO4 and Os,bipy. Only the 3'-flanking dTs were available for modification. The thymines within dTC and dTA sequences showed modification indicating that these thymines display an unstacked structure allowing both probes to attack. Under destabilizing conditions, at low ionic strength and superhelical stress, considerable unstacking was observed. We found experimental indications that under these destabilizing conditions unpaired regions might appear, probably within the dATATA sequence.

DNA, Superhelical↗
PubMed1996

Effect of flanking sequences on the right- to left-handed transition of a (dA-dT)n tract in supercoiled DNA.

Alternating (dA-dT)n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni2+ ions and high NaCl concentration (Nejedlý, K., Klysik, J. and Palecek, E., FEBS Lett. 243, 313-317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)n tract. In particular, the presence of 5'-homopyrimidine (C3) and 3'-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left-handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT)n sequence.

Base Sequence↗
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↗
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↗
PubMed1988

Site-specific chemical modification of B-Z junctions in supercoiled DNA as detected by nuclease S1 digestion, inhibition of restriction cleavage and nucleotide sequencing.

Structural distortions on the boundary between right-handed and left-handed segments in the superhelical plasmid pPK2 (a derivative of pUC19 containing (dC-dG)n segments cloned into polylinker) were studied by means of chemical probes. Strong osmium tetroxide, pyridine (Os,py) modification of DNA at native superhelical density (sigma) was found in four thymines surrounding the (dC-dG)13 segment. These results correlated with restriction cleavage inhibition (due to modification): BamHI cleavage was strongly inhibited, unlike the neighbouring XbaI and SalI (weak or no inhibition). In the (dC-dG)8 segment considerably weaker modification of the B-Z junctions was observed, accompanied by weak inhibition of BamHI cleavage, while the neighbouring SmaI and KpnI were not affected. Os,py modification of DNA at native sigma was not detected by nuclease S1 cleavage at and (dC-dG)n segment. However, this enzyme recognized and cleaved at the B-Z junction, osmium modified at more negative sigma. The results obtained with the glyoxal and diethyl pyrocarbonate modification support the idea of very narrow B-Z junctions at native sigma.

Base Sequence↗
PubMed1987

B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs.

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.

Base Composition↗
PubMed1985

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide.

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.

Binding Sites↗
PubMed1982

Changes in the cytochrome c content during the aerobic adaptation of Paracoccus denitrificans.

Concentration of cytochrome c decreases when shaking anaerobically grown cells in a non-growth medium down to 50% of the original amount in the cell, depending on the degree of aeration. Only 10-20% of this amount can be found in the adaptation medium. The main portion of cytochrome c is degraded during the adaptation. Inhibitors of proteinases do not influence the degradation. Addition of mammalian cytochrome c fully prevents the degradation of bacterial cytochrome c but not its release from cells. Potassium hexacyanoferrate(III) exhibits a similar effect as oxygen. The degradation system is probably localized in the periplasmic space of the cells.

Adaptation, Physiological↗
PubMed1977

Activities of mitochondrial enzymes during aerobic synchronous growth of aerobically and anaerobically grown Saccharomyces cerevisiae.

The mitochondrial enzymes cytochrome-c:O2-oxidoreductase (E.C.1.9.3.1), NADH:cytochrome-c-oxidoreductase (E.C.1.6.2.1), NADH: ferricyanide oxidoreductase (E.C.1.6.2.99), L-malate hydrolase (E.C.4.2.1.2) and L-malate:NADH-oxidoreductase (E.C.1.1.3.7) increase their activities during the aerobic synchronous growth of aerobically grown Saccharomyces cerevisiae in discrete steps and only once during the cell cycle. An identical phenomenon was observed during the aerobic synchronous growth of anaerobically grown yeast. The mechanism of completization of mitochondrial membranes is thus likely to be discontinuous and the same during both mitochondrial multiplication and the conversion of promitochondria to fully functioning mitochondria.

Aerobiosis↗
PubMed1977

Cardiolipin synthesis during the cell cycle of the yeast Saccharomyces cervisiae.

Cardiolipin synthesis was studied during the aerobic synchronous growth of aerobically grown yeast Saccharomyces cerevisiae. The time course of the synthesis was stepwise and the rise in cardiolipin level in cells coincided in time with the increase in cytochrome oxidase activity. This finding supports the notion of discontinuous completion of the inner mitochondrial membrane and hints at a close relation between cardiolipin and cytochrome oxidase activity.

Aerobiosis↗