Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

Nucleic acid detection using G-quadruplex amplification methodologies.

In the last decade, there has been an explosion in the use of G-quadruplex labels to detect various analytes, including DNA/RNA, proteins, metals and other metabolites. In this review, we focus on strategies for the detection of nucleic acids, using G-quadruplexes as detection labels or as enzyme labels that amplify detection signals. Methods to detect other analytes are briefly mentioned. We highlight various strategies, including split G-quadruplex, hemin-G-quadruplex conjugates, molecular beacon G-quadruplex or inhibited G-quadruplex probes. The tandem use of G-quadruplex labels with various DNA-modifying enzymes, such as polymerases (used for rolling circle amplification), exonucleases and endonucleases, is also discussed. Some of the detection modalities that are discussed in this review include fluorescence, colorimetric, chemiluminescence, and electrochemical methods.

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe.

N'-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye.

Homopolymer tract length dependent enrichments in functional regions of 27 eukaryotes and their novel dependence on the organism DNA (G+C)% composition.

BACKGROUND: DNA homopolymer tracts, poly(dA).poly(dT) and poly(dG).poly(dC), are the simplest of simple sequence repeats. Homopolymer tracts have been systematically examined in the coding, intron and flanking regions of a limited number of eukaryotes. As the number of DNA sequences publicly available increases, the representation (over and under) of homopolymer tracts of different lengths in these regions of different genomes can be compared. RESULTS: We carried out a survey of the extent of homopolymer tract over-representation (enrichment) and over-proportional length distribution (above expected length) primarily in the single gene documents, but including some whole chromosomes of 27 eukaryotics across the (G+C)% composition range from 20 - 60%. A total of 5.2 x 10(7) bases from 15,560 cleaned (redundancy removed) sequence documents were analyzed. Calculated frequencies of non-overlapping long homopolymer tracts were found over-represented in non-coding sequences of eukaryotes. Long poly(dA).poly(dT) tracts demonstrated an exponential increase with tract length compared to predicted frequencies. A novel negative slope was observed for all eukaryotes between their (G+C)% composition and the threshold length N where poly(dA).poly(dT) tracts exhibited over-representation and a corresponding positive slope was observed for poly(dG).poly(dC) tracts. Tract size thresholds where over-representation of tracts in different eukaryotes began to occur was between 4 - 11 bp depending upon the organism (G+C)% composition. The higher the GC%, the lower the threshold N value was for poly(dA).poly(dT) tracts, meaning that the over-representation happens at relatively lower tract length in more GC-rich surrounding sequence. We also observed a novel relationship between the highest over-representations, as well as lengths of homopolymer tracts in excess of their random occurrence expected maximum lengths. CONCLUSIONS: We discuss how our novel tract over-representation observations can be accounted for by a few models. A likely model for poly(dA).poly(dT) tract over-representation involves the known insertion into genomes of DNA synthesized from retroviral mRNAs containing 3' polyA tails. A proposed model that can account for a number of our observed results, concerns the origin of the isochore nature of eukaryotic genomes via a non-equilibrium GC% dependent mutation rate mechanism. Our data also suggest that tract lengthening via slip strand replication is not governed by a simple thermodynamic loop energy model.

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)].

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.

Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles.

A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.

Characterization and quantitation of the active polynucleotide fraction (PDRN) from human placenta, a tissue repair stimulating agent.

The polydeoxyribonucleotide (PDRN) fraction is an extract which forms the active component in a new formulation of the drug Placentex (a tissue repair stimulating agent), obtained from human placenta through an original proprietory extraction method. From a comparison of the UV, NMR and IR spectra of this fraction (before and after nuclease treatment) with that of a similar standard (Sigma D1501), it was shown that the active substances in the PDRN fraction mainly consist of a mixture of DNA fragments. By gel electrophoresis, the molecular weights of the DNA fragments were shown to range from 50 to 2000 base pairs. Finally, an HPLC method is described, based on an anion-exchange material capable of determining the amount of PDRN in different batches of the extract, which varied from 80 to 90%.

Characterization of a new monoclonal antibody to triplex DNA and immunofluorescent staining of mammalian chromosomes.

A monoclonal antibody, Jel 466, was prepared from mice immunized with poly[d(Tm5C)].poly[d(GA)]. The binding of Jel 466 to nucleic acids was characterized by solid phase radioimmunoassays and competition experiments. There was no binding to single-stranded DNAs or to duplexes which could not form triplexes. In addition, the antibody preferred the triplex form of poly[d(TC)].poly[d(GA)]; it bound weakly to the triplex derived from poly[d(G)].poly[d(C)], but there was no interaction with poly[d(T)].poly[d(A)].poly[d(T)]. This pattern of specificity is very different from that of Jel 318, a triplex-specific antibody that will bind to poly[d(T)].poly[d(A)].poly[d(T)]. The amino acid sequence of Jel 466 also showed very little homology with Jel 318, although both contain many positively charged amino acids. The immunofluorescent staining of mouse and human chromosomes with Jel 466 was studied. In all cases, there was a marked reciprocal relationship between the pattern of Jel 466 on the one hand and that of Hoechst 33258 and Jel 318 on the other. Jel 466 was negative for C-band and G-band but positive for R-band, whereas the opposite was found for Hoechst and Jel 318. Since C and G-bands are AT-rich and R-bands are GC-rich, these staining patterns match the sequence preferences of the two antibodies. Thus the base composition of triplex-forming DNA differs from domain to domain.

Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI2] release from the vessel wall. We have previously shown the presence of specific binding sites with a Kd of 4.2 micrograms/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinuous sucrose gradient and measuring the distribution of radioactivity. 5'-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.

Changes in chromatin and nucleic acids in rat tissues after two-week spaceflight.

The quantitative changes in nucleic acids and chromatin breakdown were followed in blood, thymus and spleen in rats after 14 day flights on board the biosatellites Cosmos-1887 and Cosmos-2044. Quantitative nucleic acid changes within 8-11 h after landing were only mild, most statistically non-significant. An analysis at 48 h after landing showed a marked decrease in a total content of DNA and RNA in spleen and thymus. Within 8-11 h after landing, the symptoms of chromatin breakdown were found as is seen in an increased concentration of its fragments-polydeoxyribonucleotides. The obtained results show that a partial adaptation to microgravity occurs up to flight day 14 in lymphoid organs. Adaptation is accompanied with a reappearing of the sensitive cells. Their chromatin breaks down, then, in a final phase of flight due to hypergravity stress manifesting itself by a temporary increase in polydeoxyribonucleotide concentration several hours after landing. The results are discussed in relation to the changes in chosen parameters after shorter or more prolonged flights.

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides.

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.

DNA-induced distamycin A fluorescence.

The fluorescent properties of the antibiotic distamycin A were investigated in a range of materials including Trypanosoma cruzi epimastigotes, chicken erythrocytes, calf thymus DNA and synthetic polynucleotides using both microscopic and spectroscopic techniques. A bright blue-white fluorescence was observed from kinetoplast DNA and chromatin after treatment with distamycin A under ultraviolet (365 nm) excitation. Considerable enhancement of distamycin A fluorescence (emission peak at 455 nm under 320-340 nm excitation) was found in the presence of DNA and poly(dA-dT).poly(dA-dT). We discuss a possible explanation for this unexpected fluorescent emission, as well as its implications for microscopic and fluorimetric studies.

Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+.

The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).

Immunochemical approach for the characterization of DNA damages induced by chemical carcinogens.

Mouse monoclonal antibody was elicited with 4-nitroquinoline 1-oxide (4NQO) modified poly(dG-dC).poly(dG-dC) and was characterized using enzyme-linked immunosorbent assay and radioimmunoassay. The antibody reacted specifically for 4NQO-poly(dG-dC).poly(dG-dC) but not for 4NQO modified DNA and synthetic polynucleotides such as poly(dG).poly(dC). The antibody crossreacted slightly with brominated or N-acetoxy-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) known to adopt Z-conformation. The antibody may recognize unique conformational change in poly(dG-dC).poly(dG-dC) modified by 4NQO. The antibody should be useful for the detection of conformational change in DNA induced by chemical carcinogens.

[An improved chemico-enzymatic method of synthesizing long DNA segments]. / Usovershenstvovannyi khimiko-fermentativnyi metod sinteza protiazhennykh fragmentov DNK.

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.

[Enzyme immunologic method for the study of left-handed DNA]. / Enzimoimmunologicheskii metod dlia izucheniia levovrashchaiushchikh DNK.

A brominated poly[d(G-C)].poly[d(G-C)] which forms a stable Z-DNA helix under physiological salt conditions was prepared. The rabbits were immunized with the brominated polynucleotide complexed with methylated bovine serum albumin. Antisera that are highly specific to the Z-DNA were produced: there is practically no interaction between the antisera and the native or denaturated DNA and the B-form of poly[d(G-C)].poly[d(G-C)]. This makes possible their use as reagents for determining the presence of Z-DNA in biological systems. A sensitive enzyme-linked immunosorbent assay (ELISA) that permits detection of 5 ng/ml Z-DNA was developed. This method was used for studying the B-Z transition and for antigenic determinant characterization. It was established, that formaldehyde amino-derivatives interact with the antigenic determinant and prevent the immunochemical assay of Z-DNA. The H1 and H3 histones prevent and and spermine increases the interaction of Z-DNA with antibodies.

[Structure of poly(dA).poly(dT) from data of x-ray diffraction, energy calculations and nuclear magnetic resonance]. / Struktura poli(dA).poli(dT) po dannym rentgenovskoi difraktsii, énergeticheskikh rashetov i iadernogo magnitnogo rezonansa.

The results of X-ray diffraction studies of poly(dA).poly(dT) have been compared with the results of energy optimization and with the NMR data in solution. Slight refinement of the X-ray and energetically optimal models leads to a very good quantitative agreement with the NMR data, that suggests similarity of the poly(dA).poly(dT) structure in a condensed state and in solution. One of the features distinguishing these models from the classic B form is a narrowed minor groove of the double helix. The anomalous properties of DNA with this sequence can be related specific organization of the water molecules near the polynucleotide.

Influence of the base sequence on the conformational behaviour of DNA polynucleotides in solution.

NMR studies were carried out on samples of the non-self-complementary tetramers d(C-A-C-A), d(T-G-T-G), d(G-A-G-A) and d(T-C-T-C) and of 1:1 mixtures of the complementary tetramers d(C-A-C-A) X d(T-G-T-G) and d(G-A-G-A) X d(T-C-T-C) at two DNA concentrations and of the self-complementary octamers d(C-A-C-A-T-G-T-G) and d(G-A-G-A-T-C-T-C). Assignments, based upon one-dimensional NOE and homonuclear-decoupling and two-dimensional correlated and NOE spectroscopies are given of the resonances of most of the base and sugar protons. Chemical shift vs temperature profiles, constructed for all samples, yielded insight into the temperature- and concentration-dependent conformational behaviour of the compounds and were used to obtain thermodynamic parameters pertaining to the stacked-single-strand----random-coil and duplex----random-coil equilibria. Vicinal proton-proton couplings were analyzed in terms of the conformation of the deoxyribose rings in the single-stranded tetramers and duplexed octamers. The NOE patterns, chemical shift profiles, imino-proton resonances and coupling data revealed that the compounds adopt B-DNA-like structures. The ratio duplexed/stacked-single-strand/random coil depends upon external conditions as well as upon base sequence. The thermodynamic data indicate that: in terms of single-helical stacking, the R-R steps (Tm 321-328 K) appear more stable than the Y-R or R-Y steps (Tm 308-316 K) and the Y-Y steps score least (Tm 290-300 K), and the duplexes consisting of alternating, d(Y-R)n, strands are more stable, in terms of delta H degrees, compared to the d(R-R)n X d(Y-Y)n duplexes. The analyses of the couplings demonstrated that the sugars of the single-stranded tetramers and duplexed octamers occur as a blend of N- and S-type conformers, with a preference for the S-type (C2'-endo) sugar conformation: upon duplex formation, no significant shift in the N-type/S-type ratio was observed. The fraction S-type sugar conformation of a given residue, %S, in the stacked-single strands was found to depend upon the nature of its own base and that of the adjacent residues: sugars in an R-R stretch display high values of %S (90-100), whereas those in Y-Y stretches show relatively low values (approximately equal to 65).(ABSTRACT TRUNCATED AT 400 WORDS)