Biomedical subjects
Publications and source records attributed to R Téoule.
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.
The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.
Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.
We report relaxation time measurements by semi-selective and totally selective NMR techniques on the thymidine imino protons of d(GGATATCC) and d(GGm6ATATCC). For these oligonucleotides helix fraying, rather than single base pair opening, is the major exchange mechanism even 25 degrees C below the Tm. We have therefore applied a new saturation transfer technique to measure exchange rates at temperatures where fraying has a very small or negligible contribution. Measurements of exchange rates as a function of temperature give significantly different activation energies for base pairs 3 and 4 in d(GGATATCC). Adenine methylation results in a slowing down of the opening rate for the m6A-T base pair but surprisingly has an even greater effect upon the adjacent non-methylated A-T base pair.
The conformation of two hexanucleotides, d(GGATCC) and d(GGm6ATCC), has been studied by proton nuclear magnetic resonance. Nuclear Overhauser effect (NOE) measurements on d(GGATCC) are in agreement with a normal B form right-handed helical structure. The single- and double-strand resonances are in fast exchange on a proton NMR time scale. The exchange is observed to be slow for d(GGm6ATCC); up to the Tm, separate resonances are observed for each state, though above the Tm exchange becomes more rapid. The preferred orientation of the adenosine methylamino group (methyl cis to N1) hinders base-pair formation. At 0 degree C irradiation of the m6A-T imino proton gives an NOE to AH2, showing that base pairing is Watson-Crick. Intra- and interresidue NOEs show that the helix is right handed and in the B form. Comparing results on the two oligomers demonstrates that adenosine methylation induces little or no change in the conformation of the helix but reduces the Tm from 45 to 32 degrees C. All of the amino proton resonances, as well as the imino resonances, have been assigned. From NOE experiments on the unmethylated oligomer we have located the Watson-Crick and non-Watson-Crick adenosine amino protons. At 0 degree C these resonances show broadening due to rotation of the amino group, and their rotation is slightly slower than for the adjacent guanosine amino group, though both these amino groups have lifetimes of less than 10 ms at 0 degree C. The imino protons show normal behavior, disappearing from the spectra ca. 20 degrees C below the Tm.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
When DNA is gamma-irradiated in aerated aqueous solution, strand breaks are produced during irradiation or the next few hours. Subsequent piperidine treatment gives rise to further DNA strand ruptures at alkali-labile sites. These different types of DNA chain breaks provoked by gamma-irradiation have been studied with oligonucleotides having defined sequences. The breaks selectively developed inside the DNA chain at alkali-labile sites by piperidine treatment appeared at lower doses preferentially at guanine positions and the order G greater than A greater than T greater than or equal to C was observed. The total contribution of the direct DNA chain ruptures, formed during irradiation and the next few hours, and those obtained by piperidine treatment was studied at doses ranging from 10 to 120 Gy. The chain breaks appeared preferentially at thymine positions and the order T greater than G greater than A greater than or equal to C was shown for the higher doses.
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.
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.
The compounds produced by gamma irradiation of deaerated aqueous solutions of thymidine have been isolated by thin-layer chromatography. The separation of (+) and (-) trans and cis diastereoisomers of thymidine "hydrates" has been carried out. Correlations between conformations given by nuclear magnetic resonance spectra and RF values are discussed.
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T-lymphocytes (T-Ly) and monocytes/macrophages are thought to be the main in vivo targets for HIV 1. We previously demonstrated, using the polymerase chain reaction (PCR), that HIV provirus could be detected in 20 out of 21 T-Ly samples and 13 out of 21 monocyte samples from HIV 1-seropositive individuals, with at least gag, env or LTR primers. In the present study, we wanted to find out whether the HIV 1 tat gene could be detected in 14 of these circulating monocyte and T-Ly samples. The tat primers were chosen in order to amplify the overall second exon of this regulatory gene. This new set of primers could not detect HIV provirus in monocytes but it did in T-Ly, among cells previously shown to be positive with one of the other 3 primer pairs. Further molecular studies should help characterize these probable monocytotropic variants and elucidate their contribution to HIV pathogenesis.
The presence of a m6A-T base pair does not give rise to a major change in helix conformation from that of a normal B DNA, but does slow down dramatically the rate of helix formation.
The synthesis of a novel phosphoramidite reagent with a hexanediol backbone is described. This reagent has been used to incorporate a phenol moiety on an oligonucleotide (ODN) directly in the course of its automated synthesis. Multiple phenol attachments can be achieved by repetitive coupling cycles. A simple and rapid immobilization method is described where phenol-modified ODNs are covalently attached to diazotized cellulose. The binding capacity of the membrane can be modulated, depending on the ODN concentration used, to ca. 180 pmol/cm2. There is at least 80% end attachment of the ODN through the phenol group. In addition, the phenol residue can be used as a carrier for the radiolabeling with 125I. The non-nucleosidic hexanediol derivative incorporated at the 5'-end of the ODN is recognized as a substrate by the T4 polynucleotide kinase and the terminal hydroxyl group is successfully phosphorylated allowing its 32P labeling.