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In a search for sequences that confer on bacterial plasmids the capacity of autonomous replication in yeast cells, we chemically synthesized polynucleotides 80 bp in length from an equimolar mixture of A and T. The random AT-polymer population, W80, was inserted into the plasmid YIp5-Kan1 (which carries the markers URA3 and G418(R), but does not replicate in yeast) and amplified in Escherichia coli. This library, representing 10 000 different AT sequences, was transformed into three species of yeast: Saccharomyces cerevisiae, Kluyveromyces lactis and Torulaspora delbrueckii. The aim was to evaluate the frequency, if any, of autonomously replicating sequences (ARSs) in the random sequences. A large number of transformants were obtained from each species. Many of them showed a stable transformed phenotype. Several W80 sequences were found many times for a given species, suggesting that each species preferred particular sequences for ARS function, although they are diverse in their primary sequence. In view of the high frequency and stability of the replicative plasmids found in the different hosts, this small random AT library may be conveniently used as a source of replicative gene vectors for genetic manipulation of many nonconventional yeast species, in place of searching for species-specific chromosomal ARSs.

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The oxazine dye (Oxazine 170) was found to induce formation of a hybrid triplex structure, poly rA:(poly dT)(2), under solution conditions in which the triplex would not otherwise form. Formation of the hybrid triplex is driven by the structural selective binding of oxazine 170 to poly rA:(poly dT)(2). Oxazine 170 serves as a lead compound for the design of new compounds that can modulate triplex formation.

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meso-Tris(4-pyridyl)[[(omega-hydroxyhexamethylene)carbamoyl]phenyl ] porphyrin was converted to its H-phosphonate derivative and conjugated using solid phase synthesis with the 5'-hydroxyl group of deoxyribonucleotides d(TCTTCCCA) and d(T)12. These conjugates were transformed into their (N-methylpyridiniumyl)porphyrin analogs in the reaction with methyl iodide. A 532 nm laser beam was utilized to photoactivate both types of the conjugates in the presence of the target 22-mer and 16-mer oligonucleotides. Photoactivation of porphyrin-oligonucleotide conjugates resulted in site-specific DNA modification characterized by a main reaction site size of approximately 5 bases.

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A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.

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We show that a double-stranded DNA segment serves as an effective template for spontaneously coupling short pyrimidine oligonucleotides containing terminal -P(O)(O-)S- and BrCH2C(O)NH- groups. The efficiency of this autoligation depends markedly on proper base-pairing between the probe oligomers and the double-stranded target. This chemistry should be useful in designing highly selective probes for double-stranded polynucleotide segments.

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A fourth capture is added to the reversible target capture procedure of the preceding paper. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the ('capture probe') which contains a 3'-poly(dA) sequence and the ('labeled probe') which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC.

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A quantitative hybridization assay termed "reversible target capture" is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe "ternary complex" is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 x 10(7) control cells.

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Oligo-heptathymidylates covalently linked to porphyrins bind to complementary sequences and can induce local damages on the target molecule. In dark reactions, iron porphyrin derivatives exhibited various chemical reactivities resulting in base oxidation, crosslinking and chain scission reactions. Reactions induced by reductants, such as ascorbic acid, dithiothreitol or mercapto-propionic acid, led to very localised reactions. A single base was the target for more than 50% of the damages. Oxidising agents such as H2O2 and its alkyl derivatives induced reactions that extended to a wider range of altered bases. The specificity of the chemical modifications observed in these systems is discussed from a mechanistic point of view.

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Synthesis of fully protected polythymidylic acids of definite lengths was carried out in solution. Decamer(T10), eicosamer(T20), tetracontamer (T40) and octacontamer(T80) were obtained in 80, 83, 75 and 52% yields respectively by a strategy of convergent synthesis.

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Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.

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The synthesis of oligothymidilic acids, (dT)m (where m = 4, 7, 10, 13, 16, 19, 22, and 25), was carried out using a solid phase approach in combination with the modified phosphotriester methodology developed in solution. Cellulose was used as the solid support after its functionalization with a specially featured dinucleoside diphosphate, 5'-0-p-chlorophenylphospho-2'(3')-0-acetyluridilyl-[2'(3')-3']-5'-0-dimethoxytritylthymidine p-chlorophenylester. The fully protected trideoxynucleoside triphosphate containing only thymidine was repeatedly used to elongate the oligonucleotide chain in the 3'-5' direction. Individual coupling yields ranged from 45% to 75%. The total time needed to prepare (dT)25 was four days. Similarly, the tridecanucleotide d(AGAAGGTACTTTT) was synthesized in good yield. The results show that this approach can be used for a fast and economic way to synthesize oligodeoxynucleotides.

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To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, we have synthesized 8-14C-labeled O6-methyldeoxyguanosine triphosphate and investigated the kinetics of its incorporation into the synthetic copolymers poly(dC'm6dG) and poly(dT,m6dG). Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5'-monophosphate with carrot phosphotransferase and then to the 5'-triphosphate via the phosphorimidazolidate formed by the action of N,N'-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was, however, higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was found to be pH dependent, and the half-life has been measured at four different pH values.

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By using anhydrous triethylamine-pyridine to selectively remove the cyanoethyl group from the fully protected oligonucleotide, a substantial improvement has been achieved in yields and the rates of condensation by the modified triester approach from the 5' leads to 3' end. The unreacted oligonucleotide containing the 5'-hydroxy group was removed by treatment with bis (triazolyl)-p-chlorophenyl phosphate after each condensation in situ. These modifications, as exemplified by the synthesis of fully protected T12, T18, T24 and T38 in 80%, 77%, 70% and 50% yields respectively, should allow the ready synthesis of polynucleotides of even longer chain lengths by purely chemical methods.

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