Biomedical subjects

D Molko

Publications and source records attributed to D Molko.

At least 19 recordsLinked to original sources

PubMed1998

Polyclonal antibodies to adenine N1-oxide: characterization and use for the measurement of DNA damage.

Adenine N1-oxide is a DNA lesion whose formation involves the specific oxidation of the adenine base by hydrogen peroxide under nonradical conditions. The damage may be measured using a HPLC/32P-postlabeling method, which however cannot be used for routine analysis. We propose herein as an alternative an immunological assay which allows a rapid evaluation of the level of adenine N1-oxide in DNA exposed to oxidative stress. Two polyclonal antibodies were raised using two different strategies for the coupling of the hapten to the protein. The first approach is based on the universal method of Erlanger and Beiser, whereas the preparation of the second antigen involves the conjugation of a morpholino derivative of adenosine N1-oxide to the carrier protein. The affinity and the specificity of those antibodies were determined by competitive enzyme-linked immunosorbent assay. The antibody obtained by the traditional method shows some cross-reactivity with normal nucleotides, whereas for the other antiserum, the selectivity was found to be higher. Therefore, this polyclonal antibody was used to quantify the level of adenine N1-oxide in calf thymus DNA oxidized either by m-chloroperbenzoic acid or by hydrogen peroxide. The detection limit of the assay is four residues of adenine N1-oxide per 10(6) normal bases. The level of adenine N1-oxide in nonmodified DNA was lower than the detection limit of the assay, whereas in mCPB- and H2O2-modified DNA, it could be up to 14 and 0.7 adenine N1-oxide residues per 10(4) normal bases, respectively.

Adenine↗

PubMed1997

Ozonolysis of 2'-deoxycytidine: isolation and identification of the main oxidation products.

The ozone-mediated oxidation of 2'-deoxycytidine (dCyd) was investigated on the basis of final product identification. The oxidation reaction gave rise to five major modified nucleosides which were isolated and characterized on the basis of extensive 1H NMR and mass spectrometry measurements. The comparison with the current knowledge of the hydroxyl radical mediated oxidation reactions of 2'-deoxycytidine in aerated aqueous solution, indicates that the formation of ozone oxidation products may be mostly explained by the opening of the pyrimidine C5-C6 double bond. Thus, the formation of the identified products obtained by ozonolysis of 2'-deoxycytidine is accounted for by the initial generation of an ozonide.

Chromatography, High Pressure Liquid↗

PubMed1995

Effects of heavy ions on nucleic acids: measurement of the damage.

In this short survey the main, available information on the molecular mechanisms of action of heavy ions on DNA is critically reviewed. Formation of single- and double-stranded DNA breaks in cells exposed to heavy particles is well established. On the other hand, base damage and, in a more general way, clustered lesions, whose formation should be increased upon exposure to heavy ions, have not yet been isolated and characterized. Efforts should be made to identify this important class of DNA damage in both isolated and cellular DNA. Sensitive and specific assays involving chemical and biochemical approaches have to be developed for such a purpose.

DNA↗

PubMed1994

Ozonolysis of thymidine: isolation and identification of the main oxidation products.

The ozone-mediated oxidation of thymidine was investigated on the basis of final product identification. The oxidation reaction gave rise to five major modified nucleosides which were isolated and characterised from extensive 1H NMR and mass spectrometry studies. The comparison with the current knowledge of the hydroxyl radical-mediated oxidation reactions of thymidine in aerated aqueous solution indicates that the formation of ozone oxidation products may be mostly explained in terms of initial generation of an ozonide. Indeed, the identified products obtained by ozonolysis of thymidine resulted from the opening of the pyrimidine C5-C6 bond.

Chromatography, High Pressure Liquid↗

PubMed1993

Fluorescent postlabelling of modified DNA bases.

Postlabelling of modified DNA bases by methods other than the 32P method has attracted little attention to date. Three approaches using fluorescence have been made: (a) detection of a characteristic adduct-DNA functionality, e.g. reaction of cis-vicinal diols of PAH-DNA adducts with a boronate-containing fluorescent molecule; (b) reaction of modified nucleotides with a reactive fluorescent molecule, e.g. detection of 8-HOdG-5'-monophosphate by reaction with dansyl chloride; (c) reaction of modified DNA bases with reagents giving fluorescent products, e.g. derivatization of 7-MeGua with phenylmalondialdehyde (PhMal). Recent results in developing the latter approach are described. Reaction of a series of 7-alkylguanines, including 7-methyl-, 7-ethyl-, 7-(2-hydroxyethyl)-, 7-carboxymethyl-, 7-(2-carboxyethyl)- and 7-(2,3-dihydroxypropyl)-guanine, with PhMal gave fluorescent products showing similar spectral properties. The detection limit for fluorescence detection following reverse-phase HPLC separation was typically less than 1 pmol injected. In order to improve the sample clean-up before analysis, an antiserum has been prepared against the fluorescent derivative of 7-(2-carboxyethyl)guanine bound to carrier protein via the carboxyl group. The resulting antiserum cross-reacted with a number of PhMal derivatives.

DNA↗

PubMed1993

Measurement of oxidative base damage to DNA by using HPLC-32P-postlabelling and GC/MS-selective ion monitoring assays.

A 32P-postlabelling assay has been developed for singling out specific oxidized base lesions. Emphasis was placed on the quantitative aspect and the accuracy of the assay, which require the use of calibration curves and microreactions, respectively. The method was successfully applied to the detection and the measurement of adenine N1-oxide and 5-hydroxymethyluracil in cells exposed to agents inducing oxidative stress including H2O2 and UV-A radiation. The sensitivity of the assay allows the detection of one lesion in 10(6) normal bases in 1 microgram of DNA. The GC/MS method when coupled to the selective ion monitoring (SIM) technique is about twenty times less sensitive, even for suitable substrates such as 5-hydroxymethyluracil and 5-hydroxyuracil, than the 32P-postlabelling assay. However, the former assay is much easier to apply, even though a derivatization step is necessary, and provides unambiguous structural information on the compound to be measured. Accurate quantitative measurements can be obtained when stable, isotopically labelled standards are available.

Chromatography, High Pressure Liquid↗

PubMed1992

Chemical synthesis of a biologically active natural tRNA with its minor bases.

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

Alanine-tRNA Ligase↗

PubMed1992

[Total chemical synthesis of natural transfer RNA].

New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

Base Sequence↗

PubMed1989

Solid phase synthesis of the 5'-half of the initiator t-RNA from B. subtilis.

Using cyanoethyldiisopropylamino phosphoramidite chemistry, four oligonucleotides constituting a part of the sequence of the initiator t-RNA from B. subtilis were synthesized. For the protection of the exocyclic amino functions of bases, phenoxyacetyl group was used for adenine and guanine, and acetyl group was preferred for cytosine. With these labile groups, final deprotection of the oligonucleotides can be performed in milder conditions, allowing the incorporation of 5,6-dihydrouridine in a 35-mer constituting the 5'-end of the t-RNA.

Bacillus subtilis↗

PubMed1989

Labile protecting groups in tRNA synthesis.

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.

Bacillus subtilis↗

PubMed1989

Synthesis of DNA fragments bearing 7,8-dihydro-8-oxo-adenine: a gamma radiation product.

In order to investigate the mutagenic effects and the repair of DNA lesions induced by gamma rays, 7,8-dihydro-8-oxo-adenine was introduced in DNA fragments using cyanoethyl phosphoroamidite method. The modified oligonucleotides were purified by HPLC and the presence of the defect in the final product was confirmed by DNA sequencing, enzymatic hydrolysis and FAB mass spectrometry.

Adenine↗

PubMed1988

Synthesis of DNA fragments containing 5,6-dihydrothymine, a major product of thymine gamma radiolysis.

5,6-Dihydrothymine is one of the most important products of base damage by gamma irradiation of DNA in anoxic conditions. This modified base is unstable in the deprotection conditions used for classical synthesis of oligonucleotides. For its incorporation in synthetic DNA fragments, a new set of amino protecting groups has been developed. The 5,6-dihydrothymidine phosphoramidite was successfully employed for the synthesis of two 14-mers and one 17-mer bearing this defect at positions corresponding to restriction enzymes sites. The presence of the modified base still intact in the oligonucleotides was evidenced by mass spectrometry in pyrolytic conditions.

DNA↗

PubMed1987

The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups.

Phenoxyacetyl (pac) and methoxyacetyl (mac) for adenine and guanine, isobutyryl for cytosine, were successfully applied as amino protecting groups both in phosphotriester and phosphoramidite approaches. As shown by N.M.R. and H.P.L.C. analysis, they are completely deblocked in less than four hours in 29% ammonia at room temperature allowing the preparation of modified DNA containing alkali labile bases such as saturated pyrimidines. The stability of N6-phenoxyacetyl-deoxyadenosine versus depurination in acidic conditions used in the detritylation step was favorably compared with that of the classic N6-benzoyl protected adenine.

Ammonia↗

PubMed1985

Influence of uracil defect on DNA structure: 1H NMR investigation at 500 MHz.

The local structure of two self complementary oligonucleotides d(GTAC-GTAC) and d(GTACGUAC) which differ only by the presence of uracil, not a normal component of DNA, have been investigated by 1H NMR at 500 MHz. The two octamers exhibit the same thermodynamical constants (t 1/2, delta H), their exchangeable protons broaden and disappear at the same temperature. The T-U substitution did not induce any significant changes on non exchangeable protons resonances from 2-D COSY and 2-D NOESY experiments. So the two octamers exhibit the same global structure. The only variation was detected by 1D NOE measurements: the base orientations around the N glycosidic bonds (chi angles) are different.

DNA↗

PubMed1985

Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases.

Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one. Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes. At the other central positions, uracil was eliminated with different initial velocities. Single stranded phi X 174 DNA fragments were used to study the influence of the sequence. Cytosine bases were deaminated to give uracil by bisulfite treatment. It was shown that the initial excision velocity of two vicinal uracil residues was decreased. The same observation was made for two uracils separated by one base. A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.

Base Sequence↗

PubMed1980

Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence.

A single-point mutation, consisting of a T-to-G transversion, was made in the third nucleotide of the conalbumin gene T-A-T-A-A-A-A homology sequence (the T-A-T-A or Goldberg-Hogness box). In an in vitro system, specific transcription of the mutant DNA was drastically decreased compared to the normal gene. This down-mutation is consistent with the idea that the T-A-T-A box is an important element for specific initiation of transcription.

Animals↗

PubMed1980

A novel class of condensing reagents in phosphodiester oligodeoxyribonucleotides synthesis. Application of the constituents of free terminal carboxy oxytocine gene.

Benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium salts have been used as novel condensing agents to promote internucleotide bond formation in phosphotriester oligodeoxyribonucleotide synthesis. The effectiveness of these stable compounds has been shown by the synthesis of the six oligodeoxyribonucleotide building blocks of the carboxy terminal oxytocine gene. Some modifications incorporated in the phosphotriester method include a rapid procedure to prepare the fully protected dideoxyribonucleotide blocks and size exclusion chromatography of the deprotected oligodeoxyribonucleotides. The triester oligodeoxyribonucleotides were studied by chemical ionization mass spectrometry.

Base Sequence↗

PubMedDate not supplied

[Synthesis of a DNA fragment containing dihydro-5,6 thymine].

The preparation of a pentadecanucleoside tetradecaphosphate containing the modified base 5,6-dihydrothymine (DHT) is reported herein. The synthesis was performed with a mixture of diastereoisomers (5R and 5S), obtained by catalytic hydrogenation of thymidine. The phosphorylated protected monomer was characterized by proton NMR and FAB mass spectrometry. It was introduced into the DNA fragment (poly dT) by the solid phase phosphotriester approach. After deprotection, the determination of the site of the modified base in the chain was made using different methods. The classical chemical sequencing method of Maxam and Gilbert showed no difference with respect to thymine moieties in the pentadecamer. To characterize the presence and the location of the modified base DHT, the 32P 5'-end labelled pentadecamer was heated in formic acid at 90 degrees C and length separated by polyacrylamide gel electrophoresis. The Electronic Impact mass spectrometry under pyrolytic conditions of the 15-mer showed that the modified base was present in the deprotected DNA fragment.

Base Sequence↗