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At least 127 records · Page 7Linked to original sources
PubMed1999

Studies on the adduct heterogeneity of benzo[a]pyrene 7, 8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides by induced circular dichroism and light absorption spectroscopy.

The binding conformations of single anti- and syn-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD) and light absorption spectroscopy. The sign of the ICD in single-stranded oligonucleotide adducts correlates with the absolute configuration of the cyclohexyl moiety of the BPDE. Adducts in oligonucleotide duplexes with UV lambdamax <350 nm exhibiting a significant duplex-induced positive ICD should have a minor groove location as the predominant conformation. Those with UV lambdamax >350 nm exhibiting either positive or negative contributions to the ICD should have intercalated binding as the predominant conformation. The magnitude of the ICD is dependent on the sequence context of the adducted strand and the particular BPDE-adduct isomer under study. In some cases, the results suggest structural heterogeneity. For instance, the (+)- and the (-)-trans-anti-BPDE-N2-dG adducts in duplexes where a dT flanks the lesion site exhibit weak positive ICD or negative ICD. These results reflect a bimodal conformational adduct distribution with contributions from both externally and internally located adducts. A key observation for the (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts is that the near-UV absorption spectra showed distinct bands corresponding to minor groove binding (lambdamax congruent with 346 nm) as well as intercalative binding (lambdamax congruent with 354 nm). Evidence for an equilibrium between the different modes of localization is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts, respectively.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Ras inhibits Jun-activated human atrial natriuretic peptide gene transcription in cultured ventricular myocytes.

p21ras is a potent regulator of myogenic cell growth and differentiation. It has been implicated as playing a major role in the genesis of cardiac hypertrophy. We examined the effect of Ras overexpression on human atrial natriuretic peptide (hANP) gene expression, a marker of hypertrophy, in neonatal rat ventricular myocytes. Transient transfection of Haras, which expresses an activated form of p21ras, effected a modest stimulation of basal hANP-chloramphenicol acetyl transferase (hANPCAT) expression. Noteworthy, the same construct inhibited both c-Jun- and Jun B-stimulated hANPCAT activity (60% and 80%, respectively). Cotransfection of a dominant-negative Ras mutant reversed this inhibition completely. The inhibitory effect was promoter selective in this system. Of those tested, only the hANP and cardiac troponin T promoters were suppressed by Ras. The inhibitory effect appears to operate through a Ras-mediated increase in c-Fos activity as evidenced by (1) the absence of additivity of the Ha-Ras- and c-Fos-mediated inhibition at higher levels of proto-oncogene expression, (2) Ras-dependent activation of c-fos gene transcription, inferred from the induction of a c-fos chloramphenicol acetyl transferase reporter (3.4-fold), and (3) reversal of Ras inhibition by a c-fos antisense oligonucleotide but not by a scrambled DNA sequence of identical base composition or the complementary sense oligonucleotide. Our findings suggest that p21tas can exert a wide range of effects on the phenotype of the cardiac ventricular myocyte. The direction that these effects take appears largely to be a function of the preexisting activation state of the cell.

Animals↗
PubMed1992

Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U937). Oligonucleotides HIV31 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-3') and HIV38 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-3') were designed to interact with the transcription initiation site (-16 to + 13) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a 3' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+3-3') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The 3' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV31 or HIV38 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U937, was treated with 10 microM HIV38. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to alpha-interferon induction resulting from oligonucleotide treatment. Both HIV31 and HIV38 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.

Base Sequence↗
PubMed1997

[Optimization of conditions for ion-pair HPLC of oligonucleotides].

Regularities of ion-pair HPLC of oligonucleotides were studied. An algorithm was suggested for the calculation of the acetonitrile gradient providing proportionality between the number of nucleotide residues and the retention time of the oligonucleotide (tR) (equidistant separation of oligonucleotides). The algorithm is based on extrapolation of the results of separation of a test mixture of five oligonucleotides varying in length from 10 to 30 units. It was shown that the logarithmic gradient of acetonitrile C0 + m ln(t) leads to the equidistant separation of oligonucleotides. The separation is characterized by two measured parameters: the retention time step and the extent of equidistantness. A functional dependence of the measured parameters on two varying parameters of the logarithmic function (C0 and m) was established experimentally. A method for calculating the expected retention time of an oligonucleotide from its nucleoside composition was suggested.

Acetonitriles↗
PubMed1976

DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides.

The quenching of the intrinsic tryptophan fluorescence of T4-coded gene 32-protein on binding to nucleotide ligands, which was described in the preceding paper, is here exploited to measure thermodynamic parameters of the single-stranded nucleic acid-gene 32-protein interaction. It is shown that binding of small ligands follows a single site binding isotherm, with association constants increasing from approximately 20 M-1 for phosphate, to approximately 10(3) M for ribose or deoxyribose 5'-phosphate, to approximately 10(4) M-1 for mononucleotides, and to approximately 10(5) M-1 for dinucleoside monophosphates (all in 0.1 M Na+). The measured binding constants appear to be about the same for homologous ribose- and deoxyribose-containing ligands and to be independent of oligonucleotide base sequence and composition. Furthermore, beyond the dinucleotide level and up to octanucleotides, the increase in binding constant with increasing chain length is only about that expected from the statistical factor resulting from the increased number of ways a longer oligonucleotide can form a protein complex. This suggests that the basic binding unit involved in gene 32-protein associations with single-stranded nucleic acids can be approximated by a dinucleoside monophosphate. Oligonucleotides long enough to accomodate two or more protein monomers are characterized by much larger association constants, indicating that binding is cooperative in protein concentration. A cooperativity parameter (omegac) of approximately 10(3) is estimated from these data, in good agreement with that deduced from the application of ligand-perturbed helix in equilibrium coil transition calculations. Values of association constants (Kcomegac) of approximately 10(8) M-1 (in 0.1 M Na+) and site size (nc) of approximately 5 (+/-1) nucleotide residues/protein monomer are determined by the fluorescence titration technique for the cooperative binding of gene 32-protein to both poly(dA) and poly(rA); these values are also in agreement with those measured by Jensen et al. (Jensen, D.E. Kelly, R.C., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7215-7228). Possible in vivo consequences and correlations of these findings with proposed roles for gene 32-protein in replication and recombination are discussed.

Binding Sites↗
PubMed1996

Interaction of the RecA protein of Escherichia coli with single-stranded oligodeoxyribonucleotides.

The RecA protein of Escherichia coli performs a number of ATP-dependent, in vitro reactions and is a DNA-dependent ATPase. Small oligodeoxyribonucleotides were used as DNA cofactors in a kinetic analysis of the ATPase reaction. Polymers of deoxythymidilic acid as well as oligonucleotides of mixed base composition stimulated the RecA ATPase activity in a length-dependent fashion. Both the initial rate and the extent of the reaction were affected by chain length. Full activity was seen with chain lengths > or = 30 nt. Partial activity was seen with chain lengths of 15-30 nt. The lower activity of shorter oligonucleotides was not simply due to a reduced affinity for DNA, since effects of chain length on KmATP and the Hill coefficient for ATP hydrolysis were also observed. The results also suggested that single-stranded DNA secondary structure frequently affects the ATPase activity of RecA protein with oligodeoxyribonucleotides.

Adenosine Triphosphatases↗
PubMed1999

Cooperative binding of oligonucleotides to adjacent sites of single-stranded DNA: sequence composition dependence at the junction.

The quantitative parameters of cooperative binding of deoxyribooligonucleotides to adjacent sites by double helix formation have been determined as a function of sequence composition at the junction. The base stacks 5'-Py/p-Py-3', 5'-Pu/p-Py-3' and 5'-Pu/p-Pu-3' (p is phosphate group, Py and Pu are pyrimidine and purine nucleoside, respectively) including mismatches on the 3'-side of the junction were studied using complementary addressed modification titration (CAMT) at 25 degrees C and pH 7.5, 0.16 M NaCl, 0.02 M Na2HPO4, 0.1 mM EDTA. The equilibrium binding constants of alkylating derivatives of 8-mer oligonucleotides (reagents) with 22-mer oligonucleotides (targets) were determined using the dependence of the target limit modification extents on the concentrations of the reagents. The parameters of cooperativity were calculated as the ratio of binding constants of reagents in the presence and the absence of a second 8-mer oligonucleotides (effectors) occupying the adjacent site on the 22-mer targets. For the stacks 5'-Py/p-Py-3' the parameters of cooperativity were around unity both for matched and mismatched nucleotides at the junction indicating the absence of cooperativity. The parameters of cooperativity for the stacks 5'-Pu/p-Pu-3' were higher than for the stacks 5'-Pu/p-Py-3' in perfect and non-perfect duplexes. Discrimination of mismatches was higher in nicked than in normal duplexes.

Alkylation↗
PubMed1994

Computational DNA sequence analysis.

This paper reviews several new developments in computer and statistical analysis of DNA and protein sequences. We present criteria and describe means for assessing and interpreting genomic inhomogeneities within and between sequences. These include: (a) characterizations of short oligonucleotide biases and general compositional tendencies; (b) molecular evolutionary reconstructions based on dinucleotide relative abundance distance measures and partial orderings; and (c) the application of r-scan statistics, quantile distributions, and score-based analyses to identify clustering, overdispersion, and excessive evenness in the distribution of a marker array along a sequence. These apply, for example, to restriction sites, microsatellite runs, regulatory motifs, and nucleosome placements. Furthermore, (d) the definition and determination of rare and frequent oligonucleotides and peptides provides another perspective on sequence heterogeneity, and (e) score methods are also applied in exon and gene locations. Most of the ideas and methods are illustrated with respect to bacteriophage genomes, to megabase amounts of several eukaryotic sequences, to a diverse collection of bacterial sets, to mitochondrial chromosomes, and to a broad assembly of viral genomes.

Amino Acid Sequence↗
PubMed1975

Separation of pyrimidine deoxyribooligonucleotides according to length and composition using thin-layer chromatography on deae-cellulose.

A procedure has been developed for the fractionation of pyrimidine deoxyribo-oligonucleotides, PynPn+1, according to length and composition using ascending thin-layer chromatography on DEAE-cellulose. The separation of oligonucleotides according to length (n=1-7) into individual isopliths was carried out using a linear 0-0.35 M sodium chloride gradient in 0.01 M sodium acetate buffer of pH 5.1-5.3 and 5 M urea. Then the oligonucleotides of individual isopliths were separated according to composition using the same gradient of sodium chloride in 0.01 M sodium acetate or sodium citrate buffer of pH 3.2. The procedure can be used for rapid fractionation (2-3 h) and serial determination of the frequencies of pyrimidine sequences differing in length and composition in DNAs of various origin. The procedure can be used for both preparative and analytical applications. It was employed to study the distribution of 5-methylcytosine in pyrimidine isopliths of some DNAs.

Animals↗
PubMed1975

Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.

RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.

Amino Acids↗
PubMed2001

Open-tubular capillary electrochromatography of bovine beta-lactoglobulin variants A and B using an aptamer stationary phase.

DNA aptamers that form a G-quartet conformation were covalently attached to a capillary surface for open-tubular capillary electrochromatographic separation of bovine beta-lactoglobulin variants A and B, which vary by 2 of their 162 amino acid residues. Separation was achieved using a 4-plane, G-quartet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G-quartet structure, or using capillary zone electrophoresis on a bare capillary under similar experimental conditions. Separation was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer-coated and linker-only stationary phases, while in Tris buffer, retention times were almost doubled for the linker-only capillary. When Tris buffer is the mobile phase, there appears to be weaker interactions between the proteins and the stationary phase that may result in a gentler, less denaturing separation than is commonly achieved using hydrocarbon-based stationary phases.

Animals↗
PubMed1987

RNA fingerprinting using a small horizontal agarose gel electrophoresis apparatus.

The electrophoresis step in the RNA fingerprinting procedure has been modified by replacing the large and expensive electrophoresis tank with a horizontal agarose gel electrophoresis box, a common piece of laboratory equipment. Two RNA molecules (67 and 298 nucleotides long) transcribed in vitro from a truncated human beta-globin gene have been fingerprinted and analyzed by the modified technique. The resolution of the oligonucleotides in this procedure is comparable to that seen using the tank and is sufficient to separate relatively large oligonucleotides with similar base composition.

Base Sequence↗
PubMed1998

Polishable and renewable DNA hybridization biosensors.

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.

Animals↗
PubMed1996

Creating RNA bulges: cleavage of RNA in RNA/DNA duplexes by metal ion catalysis.

The manipulation of a single-stranded RNA target by forming different RNA/antisense hybrids demonstrates the possibility of cleaving the RNA strand within duplexes. This was achieved using the sequence composition of the antisense oligonucleotide, an approach that results in various bulges [unpaired base(s)] in the RNA target, which is then cleavable at these specific bulge sites under free metal ion or metal complex catalysis. RNA cleavages promoted by metal ions were performed under mild conditions and characterized by separating the RNA fragments carrying end label. The observed products result from intramolecular transesterification causing RNA strand scission. No detectable cleavage of the RNA was observed with either a fully complementary RNA/antisense hybrid or a bulged base in the antisense strand. A molecular modeling study of the RNA backbone suggests that the local conformation of the RNA backbone at a bulge in such hybrid duplexes greatly facilitates the metal-assisted catalytic cleavage. Endonucleolytic RNA cleavage within an RNA/antisense hybrid by metal complexes attached to the antisense oligonucleotide might lead to a new approach in antisense technology with artificial ribonucleases which operate with catalytic turnover.

Base Sequence↗
PubMed1979

Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods.

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Adenine↗
PubMedDate not supplied

Effect of streptococcal extracellular nuclease on the carrier activity of RNA for streptolysin S.

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

Animals↗
PubMed2003

Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA.

Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC DNA and 1.8 ns in poly-AT DNA. The distribution of excited state lifetimes of single molecules of TOTO intercalated into oligonucleotides having varying proportions of poly-GC sequences relative to poly-AT sequences were analyzed as a function of the fraction of poly-GC. By using excited state lifetime distributions from the purely GC and purely AT oligonucleotides as a basis set, it was possible to estimate the GC content of oligonucleotides with intermediate GC composition to within a few percent error. This serves as a model for the analysis of equilibrium binding distributions in DNA using single-molecule methods.

Base Sequence↗
PubMed1977

Precursors of ribosomal RNA in the cellular slime mold Dictyostelium discoideum. Isolation and characterization.

The pathway of ribosomal RNA biogenesis in Dictyostelium discoideum has been defined through identification, isolation, and characterization of the rapidly labeled nuclear RNAs which are intermediates in the process. Comparison of the methylation patterns, base compositions, two-dimensional oligonucleotide maps, and hybridization properties of these intermediate RNAs with those of mature rRNAs has established clearly the precursor-product sequence relationships supporting the following scheme for rRNA production and processing: (formula: see text) The relationship of the 37 S RNA of Dictyostelium to primary rRNA transcripts of prokaryotes and other eukaryotes is discussed.

Dictyostelium↗