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1.
Intramolecular triplex formation of the purine.purine.pyrimidine type   总被引:4,自引:0,他引:4  
F M Chen 《Biochemistry》1991,30(18):4472-4479
Six octadecamers with hairpin motifs have been synthesized and investigated for possible intramolecular triplex formation. Electrophoretic, hypochromic, and CD evidence suggest that d(CCCCTTTGGGGTTTGGGG) and d(GGGGTTTGGGGTTTCCCC) can form G.G.C intramolecular triplexes via double hairpin formation in neutral solutions, presumably with the terminal G tract folding back along the groove of the hairpin duplex. In contrast, d(GGGGTTTCCCCTTTGGGG) and the three corresponding 18-mers containing one G and two C tracts each forms a single hairpin duplex with a dangling single strand. The design of the sequences has led to the conclusion that the two G tracts are antiparallel to each other in such a triplex. Magnesium chloride titrations indicate that Mg2+ is not essential for such an intramolecular triplex formation. The main advantage of our constructs when compared to the intermolecular triplex formation is that the shorter triplex stem can be formed in a much lower DNA concentration. The merit of G.G.C triplex, in contrast to that of C+.G.C, lies in the fact that acidic condition is not required in its formation and will, thus, greatly expand our repertoire in the triplex strategy for the recognition and cleavage of duplex DNA. Spectral binding studies with actinomycin D (ACTD) and chromomycin A3 (CHR) as well as fluorescence lifetime measurements with ethidium bromide (EB) suggest that although hairpin duplexes bind these drugs quite well, the intramolecular triplexes bind poorly. Interestingly, the binding densities for the strong-binding hairpins obtained from Scatchard plots are about one ACTD molecule per oligomeric strand, whereas more than two drug molecules are found in the case of CHR, in agreement with the recent NMR studies indicating that CHR binds to DNA in the form of a dimer.  相似文献   

2.
The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.  相似文献   

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A high-resolution liquid chromatographic system was utilized to analyze urine samples of twelve nonchemical suicide victims for the relative composition of ultraviolet-absorbing compounds. The analyses demonstrated a decrease in the output of uric acid, characteristic of severe depression, and an increase in the excretion of hypoxanthine. The excretion of 7-methylxanthine was found to be variable and xanthine was normal.  相似文献   

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In the presence of azaserine an inhibitor of phosphoribosylformylglycineamidine synthetase (EC 6.3.5.3) the incorporation of [2-14C]glycine into 5′-phosphoribosylglycineamide and its formyl derivative was measured in 105,000g supernatant fraction prepared from a homogenate of adrenal cortex. Corticotropin at a level of 1-0.001 nm markedly stimulated in 10 min these early steps of purine biosynthesis. The stimulus was in addition to that achieved with added glucose-6-phosphate and NADP. Increased synthesis of precursors of purine nucleotides is due to ACTH activation of adrenal glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and thus the pentose cycle with an increase in 5′-phosphoribosylpyrophosphate. The generation of this latter compound is presumed to be a rate-limiting factor to 5′-phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) the first enzyme of de novo purine biosynthesis.  相似文献   

8.
1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.  相似文献   

9.
The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.  相似文献   

10.
The in vitro inhibition of purine biosynthesis de novo by a series of cyanopyrazoles was studied. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. GAR synthesis was inhibited at a lower degree at the same range of concentrations. The compounds demonstrated a similar pattern of inhibition of the last steps, e.g. AICAR formylation and cyclization as found on the whole pathway.  相似文献   

11.
Summary The UV induced mutation frequency of a given base pair located at different sites within the CYC1 gene of Saccharomyces cerevisiae was found to vary by more than fifty fold, indicating the existence of hotspots and coldspots typical of those found in other organisms. We were unable, however, to find any feature of the nucleotide sequence at or near the sites of mutation that explains this variability. These and other data suggest that hotspots are not located within regions particularly susceptible to the formation of premutational lesions. More probably the variation in mutability depends on differences in the activity of enzymes responsible for producing mutations, though the reasons for these differences are not understood and may depend on factors not directly related to nucleotide sequence.  相似文献   

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Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).  相似文献   

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A Debin  C Malvy    F Svinarchuk 《Nucleic acids research》1997,25(10):1965-1974
In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.  相似文献   

17.
Recently, P.A. Beal and P.B. Dervan, expanding on earlier observations by others, have established the formation of purine.purine.pyrimidine triple helices stabilized by G.GC, A.AT and T.AT base triples where the purine-rich third strand was positioned in the major groove of the Watson-Crick duplex and anti-parallel to its purine strand. The present nuclear magnetic resonance (n.m.r.) study characterizes the base triple pairing alignments and strand direction in a 31-mer deoxyoligonucleotide that intramolecularly folds to generate a 7-mer (R/Y-)n.(R+)n(Y-)n triplex with the strands linked by two T5 loops and stabilized by potential T.AT and G.GC base triples. (R and Y stand for purine and pyrimidine, respectively, while the signs establish the strand direction.) This intramolecular triplex gives well-resolved exchangeable and non-exchangeable proton spectra with Li+ as counterion in aqueous solution. These studies establish that the T1 to C7 pyrimidine and the G8 to A14 purine strands are anti-parallel to each other and align through Watson-Crick A.T and G.C pair formation. The T15 to G21 purine-rich third strand is positioned in the major groove of this duplex and pairs through Hoogsteen alignment with the purine strand to generate T.AT and G.GC triples. Several lines of evidence establish that the thymidine and guanosine bases in the T15 to G21 purine-rich third strand adopt anti glycosidic torsion angles under conditions where this strand is aligned anti-parallel to the G8 to A14 purine strand. We have also recorded imino proton n.m.r. spectra for an (R-)n.(R+)n(Y-)n triplex stabilized by G.GC and A.AT triples through intramolecular folding of a related 31-mer deoxyoligonucleotide with Li+ as counterion. The intramolecular purine.purine.pyrimidine triplexes containing unprotonated G.GC, A.AT and T.AT triples are stable at basic pH in contrast to pyrimidine.purine.pyrimidine triplexes containing protonated C+.GC and T.AT triples, which are only stable at acidic pH.  相似文献   

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Neurospora crassa can utilize purines and their metabolic products as a nitrogen source. Regulation of the five enzymes required for uric acid metabolism was studied. The first three enzymes of this catabolic pathway are controlled in a complex manner that involves both induction and repression. Both uricase and allantoicase were induced by uric acid while allantoinase was induced by either uric acid or allantoin. Synthesis of all three of these enzymes was repressed by the end product, ammonia. The ure-2 mutant, which is urease deficient and cannot derive ammonia from purines, shows a hyperinducibility of these same three enzymes. The last two enzymes of the pathway, ureidoglycollase and urease, were found to be constitutive.  相似文献   

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