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.

[Total chemical synthesis of natural transfer RNA]. / Synthèse chimique totale d'un ARN de transfert naturel.

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.

Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA.

2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.

Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine.

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.

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.

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.

Measurement of the radiosensitivity of a radiation-induced adenine defect in a single stranded DNA chain.

To determine the radiation sensitivity of an adenine defect inside the DNA chain, an oligonucleotide bearing 7,8-dihydro-8-oxoadenine was chemically synthesized by the phosphoramidite method. This damaged oligonucleotide 32P-labelled at one end was gamma irradiated in an aerated aqueous solution, and heated with piperidine. The fragments obtained were separated by polyacrylamide sequencing gel electrophoresis, according to their chain lengths. Mean radiosensitivity coefficients for the four natural nucleotides and the relative radiation sensitivity of 7,8-dihydro-8-oxoadenine leading to chain rupture in alkaline medium, could be obtained from the band intensities by computer calculation. An unexpectedly high value was obtained for the damaged adenine residue. Determinations were performed for different doses of irradiation.

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.

Gamma-irradiation of homodeoxyoligonucleotides 32P-labelled at one end: computer simulation of the chain length distribution of the radioactive fragments.

Electrophoresis on polyacrylamide gels of the fragments resulting from gamma-irradiation of single-stranded oligodeoxyribonucleotides labelled at their 5'- or 3'-end proved to be a potent tool for the analysis of the radiation-induced chain breakage of DNA. Owing to the fact that the oligonucleotide may be ruptured at more than one site, the counting of the electrophoresis bands must be corrected and it is necessary to assess the influence of the cleavage position on the band intensities. A complicating factor is the inhomogeneity of the system due to the presence of the four bases A, T, C and G. To circumvent this problem, the homooligodeoxyribonucleotides (dA)15, (dC)15, (dT)15 were used as experimental probes. They were gamma-irradiated in solution, heated in alkali and the resulting fragments separated by gel electrophoresis. A computer simulation of the band intensities was compiled based on the general assumption that the chain breakage is homogeneous. The experimental results obtained from the homooligodeoxyribonucleotides labelled at either the 5' or the 3'-end are in excellent agreement with theoretical calculations. Abacus giving the gel band intensities (percentage) against the nucleotide positions and the remaining intensity of the original oligonucleotide have been obtained.

Sites of gamma radiation-induced DNA strand breaks after alkali treatment.

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.

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.

Fluid phase activation of proenzymic C1r purified by affinity chromatography.

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.

Isobongkrekic acid, a new inhibitor of mitochondrial ADP-ATP transport: radioactive labeling and chemical and biological properties.

An isomer of bongkrekic acid, designated as isobongkrekic acid, has been isolated from ethereal extracts of Pseudomonas cocovenenans grown on defatted coconut. Isobongkrekic acid was also obtained by alkaline treatment of bongkrekic acid. Isobongkrekic acid possesses the same ultraviolet spectrum and the same molecular weight as bongkrekic acid; it has a similar infrared spectrum but not the same nuclear magnetic resonance (NMR) spectrum. The differences in NMR data were interpreted to mean that isobongkrekic acid differs from bongkrekic acid by the configuration of the dicarboxylic end; whereas the two carboxylic groups of the dicarboxylic end have the trans configuration in bongkrekic acid, they have the cis configuration in isobongkrekic acid. Differences between bongkrekic and isobongkrekic acids are lost after catalytic hydrogenation of the molecules. Isobongkrekic acid, like bongkrekic acid, is an uncompetitive inhibitor of ADP transport in mitochondria, provided the mitochondria are preincubated in the presence of the inhibitor and a minute concentration of ADP. The inhibitory and binding efficiency of isobongkrekic acid is considerably increased below pH 7. The number of high affinity sites for [3H] isobongkrekic acid is 0.13 to 0.20 nmol/mg protein in rat liver mitochondria and about 1 nmol/mg protein in rat heart mitochondria, i.e., similar to the number of high affinity sites for [3H] bongkrekic acid. Isobongkrekic and bongkrekic acids compete for the same site, but the affinity of isobongkrekic acid for mitochondria is one-half to one-fourth that of bongkrekic acid.