PubMed Health⌕ Search

Biomedical subjects

P Karlovský

Publications and source records attributed to P Karlovský.

14 recordsLinked to original sources
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↗
PubMed1988

Calculation of individual cleavage rates from partial digests in restriction endonuclease kinetics.

A correction for results obtained by an analysis of DNA molecules partially cleaved with restriction endonuclease was suggested. The correction was proved on model data. Applications to (i) electron-microscopic analysis of singly cleaved molecules, (ii) partial digestion of a circular molecule followed by complete digestion with a second enzyme, (iii) systems with great cleavage differences, and (iv) partial cleavage of end-labelled molecules were discussed.

Bacteriophage lambda↗
PubMed1987

Tn1000 insertional mutagenesis of cloned repressor gene of the phage L: plasmid oligomerization in the presence of F'lac.

An ampicillin resistance plasmid carrying the cloned repressor gene cII of the L phage (Salmonella typhimurium) was conducted by F'lac into an F- recipient. Two types of plasmids were isolated from Apr transconjugants. The majority of plasmids were dimers with one copy of Tn1000 inserted, the minority being monomers with one copy of Tn1000. This proportion remained unaltered when we used the F'lac strain transformed with a monomeric form of the recombinant plasmid as a donor. An extensive oligomerization of pBR322-originating plasmids was proved in the presence of F'lac; its presumable relationship to transposition-related processes is suggested.

Cloning, Molecular↗
PubMed1987

Osmium tetroxide recognized structural distortions at junctions between right- and left-handed DNA in a bacterial cell.

It was shown for the first time that the structural distortions at the junctions between contiguous right-handed and left-handed Z-DNA segments can be recognized in bacterial cells. E. coli containing recombinant plasmid pPK1 (a derivative of pUC19 containing (dC-dG)13 and (dC-dG)16 blocks) were treated with osmium tetroxide, 2.2'-bipyridine (Os,bipy); after this treatment pPK1 DNA was isolated by the boiling method. pPK1 DNA was then cleaved with BglI, and inhibition of BamHI (with its recognition sequence GGATCC lying on the boundary between the (dC-dG)n segments and the pUC19 nucleotide sequence) cleavage was tested. Treatment of cells with 2 mmol/l Os,bipy resulted in a strong inhibition of BamHI cleavage at both restriction sites showing a site-specific osmium modification at the B--Z junction. About the same inhibition of BamHI cleavage was observed after treatment of isolated pPK1 DNA with 0.2 mmol/l Os,bipy.

DNA Restriction Enzymes↗
PubMed1987

Transfer of Drosophila melanogaster transponsable genetic element mdg-4 into plant cells.

The copia-like element mdg-4, as a component of the Drosophila genomic fragment Dm111, was cloned into the vector pIB16. The chimaeric plasmid pIB16 [Dm111] was used to transform tobacco cells as a cointegrate with pTiC58 (Agrobacterium tumefaciens), pTiC58::pIB16 [Dm111]. The growth properties of primary crown-gall tumours were followed, and the DNA of one Nicotiana tabacum transformant was further analysed. The DNA/DNA molecular hybridization of digests of genomic DNA with 32P-labelled pDm111 probe demonstrated full-length insertion of Dm111 into N. tabacum genome. We were not able to detect any common sequence with Dm111 in untransformed tobacco cells.

Animals↗
PubMed1986

Re-evaluation of a method calculating cleavage rates at different sites of DNA from partial digestion of end-labelled molecule.

Nucleases cleave the same targets at different rates in different parts of DNA molecules. The cleavage rates can be calculated from the amounts of particular fragments occurring in the DNA partial digests, but these are very complex and difficult to separate. To simplify the analysis, the use of end-labelled DNA molecules was suggested by Lutter. It is shown in the present communication that the fundamental equation of Lutter's approach is erroneous. A correct equation is derived and recommended to be used for re-calculation of the results obtained previously.

DNA↗
PubMed1986

A re-evaluation of evidence attributing the difference in cleavage rates of restriction endonuclease at different sites in the substrate to differences in Km values.

The only result supporting the hypothesis that the differences in restriction endonuclease cleavage rates at various target sites are caused by differences in Km values was reported by Forsblom, Rigler, Ehrenberg, Petterson & Philipson [(1976) Nucleic Acids Res. 3, 3255-3269]. The present work shows that the kinetic analysis in that paper is based on incorrect derivation and in fact provides no support for the hypothesis mentioned.

Binding Sites↗
PubMed1986

Kinetics of circular DNA molecule digestion by restriction endonuclease. Computation of kinetic constants from time dependence of fragment concentrations.

A model for kinetics of circular substrate cleavage by restriction endonuclease was formulated. The aim of the analysis of the model was to extract kinetic constants for all target sites from time-dependence of fragment concentration in reaction products. That was proved to be possible for molecules with an odd number of fragments only. A symmetry of the molecules with an even number of fragment is the cause. A solution for molecules with an odd number of fragments was found and methods for dealing with the other molecules were suggested.

DNA Restriction Enzymes↗
PubMed1986

Specific binding affinity for DNA of the L phage (Salmonella typhimurium) in extracts of Escherichia coli.

The repressor gene cII of the L phage was cloned into plasmid pHC624 and expressed in E. coli. Two separate binding affinities for L phage DNA were identified during fractionation of protein extract of that strain. The activity that salts out in low concentration of ammonium sulphate belonged to the repressor, the activity that salts out in high concentrations of (NH4)2SO4 was proved to be of E. coli origin. Binding sites for the two proteins are located on different fragments of the L phage genome.

Bacterial Proteins↗
PubMed1986

Integration of transposon Tn10 into phage L (Salmonella typhimurium).

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.

DNA Transposable Elements↗
PubMed1986

Hot spot for Tn1000 insertions in cloned repressor gene of the L phage.

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.

Bacteriophages↗
PubMed1982

Tetraphenylborate-sensitive electrode for measuring membrane potential.

The paper describes the construction of a new type of ion-selective electrode sensitive to tetraphenylborate (TPB-) and its electric characteristics. The electrode responds to increasing concentrations of the TPB- anion in accordance with the Nernst equation and can be used down to 0.1 microM concentration. The applicability of the electrode for measuring the membrane potential (positive inside) was proved in inside-out oriented membrane vesicles derived from Paracoccus denitrificans. The calculated values were 175 +/- 12 mV with NADH and 180 +/- 6 mV with succinate.

Electrodes↗