Isomerisation and conformation studies of (+)- and (-)6-hydroxy-5, 6-dihydrouridine.

(1) "Uridine hydrates" i.e. (+)- and (-)6-hydroxy-5, 6-dihydrouridine were formed under gamma irradiation in a deaerated aqueous solution of uridine. (2) The structures of two diastereoisomers were determined by spectroscopic measurements (infrared, ultraviolet and NMR) and verified by stereospecific synthesis; uridine hydrates were prepared by mild reduction of trans(+)- and (-)iodohydrins with acetic acid and zinc power. (3) The carbon 6 epimerisation of uridine hydrates 6R or 6S was performed in triated water (pH 5.5, 30 degrees C) and at the same time tritium incorporation on carbon 5 was noted. The mechanism of these reactions could be explained by the opening of the N1-C6 bond of the pyrimidine ring, followed by ketoenolisation reaction of carbons 4 and 5. (4) The 250 MHz NMR analysis has allowed us to determine the nucleoside conformations. Nucleosides had mainly the S(C2' endo) conformation. A slight preference of gauche-gauche (gg) rotamer of the exocyclic hydroxymethyl group was noted and the aglycone was in the anti conformation.

[Synthesis of a DNA fragment containing dihydro-5,6 thymine]. / Synthèse d'un fragment de DNA comportant la 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.

A proton 2D-NMR study of an oligodeoxyribonucleotide containing N6-methyladenine:d(GGm6ATATCC).

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.

Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity. In normal rat, AVP mRNA can be detected in magnocellular neurons of the supraoptic and paraventricular nuclei and in parvocellular neurons of the suprachiasmatic nucleus. AVP mRNA was present throughout the cytoplasm of the cell bodies, their processes, and in punctate structures in the vicinity of the AVP cell bodies. Most neurons containing AVP mRNA also contain AVP immunoreactivity, but the staining intensity was not consistently correlated for each reaction. A few neurons contained AVP mRNA without detectable AVP immunoreactivity. In the Brattleboro rat, staining intensity of the reaction was lower than in normal rat and the AVP mRNA was restricted mostly to the periphery of the cytoplasm. In this strain, the neurons containing the AVP mRNA did not contain AVP or oxytocin immunoreactivity. These results demonstrate that neuropeptide mRNA can be detected in semi-thin sections with a biotinylated oligonucleotide probe, and that AVP gene deletion provokes modification of the intracellular localization of the AVP mRNA.

Fast solid support detection of human papillomavirus in in vitro amplified DNA using a DNP-anti DNP monoclonal antibody couple.

In some anogenital lesions the detection of certain types of human papilloma virus, especially oncogenic types, is of interest. In a first step during a prospective study, we compared two methods for the detection of human papillomavirus (HPV) DNA in clinical samples: Southern blotting followed by hybridization with a cloned radioactive genomic probe and a classical polymerase chain reaction (PCR) followed by hybridization with a 32P-labelled oligonucleotide probe. 118 biopsies and swabs were examined for HPV 6/11, 16, 18 and 33, 67 positive reactions were found by both methods, 5 positives only by PCR and 2 positives only by Southern blot for unidentified HPV. Patients with anogenital condylomas, dysplasias and carcinomas or asymptomatic patients were studied. Most high grade (II and III) dysplasias were associated with HPV 16 and HPV 18. Condylomata lesions and low grade dysplasia (grade I) were associated mostly with HPV 6/11, mixed type of HPV, less frequently with HPV 16 or HPV 18. As a second step a nested PCR coupled to solid support detection method was used as described by Sauvaigo et al. (1990) Nucleic Acids Res. 18, 3175-3183) to study a panel of 30 previously qualified different HPV DNA extracts. In this procedure the second round of PCR amplification involves biotinylated and dinitrophenylated labelled primers allowing the capture of PCR amplified HPV DNA sequences on streptavidin coated tubes and its revelation. We describe an improvement of HPV DNA detection by means of single-step immunoenzymatic revelation involving anti-DNP monoclonal antibodies conjugated to horseradish peroxidase enzyme. A perfect correlation with the previous results was obtained. This solid support method allows a faster and easier HPV typing compared to methods using membrane transfer.

DNA strand breaks induced by gamma irradiation and alkaline treatment: a computer program for the analysis of the influence of the sequence.

Deoxyoligonucleotides 32P-labelled at one end have been used to probe the DNA chain breakage induced by external gamma-irradiation and alkali treatment. The fragments were separated according to their chain lengths by polyacrylamide gel electrophoresis and their radioactivity counted. Quantitative analysis of the distribution pattern of the intensity fragments was performed by computer. In this article the principles of the calculation program are given. The basic units corresponding to adenine, thymine, cytosine and guanine exhibited different radiosensitivities. It was possible to estimate the mean chain rupture per nucleotide and per Gray for G, T, C and A. These coefficients were derived from experimental values by iteration. Thus, it is possible to simulate very accurately the oligonucleotide fragment intensities. The relative error was usually less than 10 per cent for most of the nucleotide units. In this way small differences in the band intensities may be demonstrated. They can be explained by variations of the local conformation of the biopolymer.

Radiation-induced DNA damage and its repair.

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.

Comparison of radiolysis products of thymine and thymidine with e.s.r. results.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.

[Separation of the four diastereoisomers of 6-hydroxy-5, 6-dihydrothymidine formed by gama-radiolysis of thymidine in deaerateed aqueous solution (author's transl)]. / Séparation des quatre diastéréoisoméres de l'hydroxy-6-dihydro-5,6-thymidine formés par radiolyse gamma de la thymidine en solution aqueuse désaérée

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.

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.

Synthesis and spectroscopic properties of two classes of 5,6-dihydrothymidine derivatives. Action on the Ehrlich's ascites cells thymidine kinase.

Trans (+) and (-) 6-alkoxy-5-bromo-5,6 dihydrothymidine and trans (+) and (-) 6-alkoyloxy-5-bromo-5,6-dihydrothymidine compounds have been prepared. The synthesis of these substances (alkoxy : methoxy, ethoxy, butyloxy and isoamyloxy and alkoyloxy : acetoxy and bensovloxy) is described. Diastereoisomers of all products have been isolated by thin layer chromatography and their spectroscopic properties (IR, UV, NMR, mass spectrometry) studied. These compounds have been shown to be competitive inhibitors of Ehrlich's ascites cells thymidine kinase with respect ot thymidine.