Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: Modeling protein-DNA complexes with dyad symmetry and known protein structures

A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters—the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts—are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts. © 1996 Wiley-Liss, Inc.     

Effects of sodium azide on replicative and repair DNA synthesis in barley embryos

Sodium azide inhibited protein synthesis and replicative DNA synthesis at doses used for mutation induction (0.1–1 mM) in barley embryos. During subsequent treatment for 24 h using lower doses of azide, partial recovery from this inhibition was observed, especially in the case of replicative DNA synthesis. Contrary to expectation, cysteine acted as a weak enhancer of the inhibitory effect of azide on DNA replication. Under conditions of minimal replicative DNA synthesis, DNA-repair synthesis was detected after the azide action as measured by the BND-cellulose method. Benazzmide — an inhibitor of poly(ADP-ribose)-polymerase — stimulated the azide-dependent repair synthesis approximately 2-fold.     

A rapid assay for cellular deoxyribonucleic acid

A method is described for the rapid and specific extraction and subsequent fluorometric assay of DNA from less than 2 × 10⁴ diploid mammallan cells—0.1 μg of DNA. The procedure can be routinely completed in 3 hr and can be applied to quantitation of [³H]thymidine incorporation into these small quantities of DNA.     

Dynamics of kinks in inhomogeneous polynucleotide chains

In the present paper the dynamics of the nonlinear conformational excitations — kinks, in inhomogeneous polynucleotide DNA chains is investigated. To calculate the kink rest energy E₀ and its size d the method of dynamical interval is used. This makes it possible to take into account that all coefficients of the model dynamic equation — the sine-Gordon equation depend on the sequence of bases. It is shown that the method gives an opportunity to calculate dynamical characteristics of any artificial and real sequences that is important for implementing tasks associated with the search and analysis of functionally important DNA sites.     

Diagnostics of ataxia-telangiectasia by an express test based on the indirect immunofluorescence method

Ataxia-telangiectasia (AT) is a severe hereditary neurodegenerative disease developing in the presence of mutations in both alleles of the gene atm. This gene encodes the key protein of cell response to DNA damage—ATM protein kinase. During the appearance of double-strand DNA breaks, the ATM protein kinase is autophosphorylated and its active form appears in the cell—phospho-ATM (P-ATM)—revealed by a modified method of indirect immunofluorescence. In nuclei of cells containing the normal gene atm, after the effect of agents producing double-strand DNA breaks, P-ATM is detected, whereas in the cells carrying mutant variants of the gene atm P-ATM is not found. This peculiarity can be used in clinics for confirmation of diagnosis of AT in complicated cases.     

A method for detecting protein-DNA interactions at sites of chromatin replication

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-³²P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.     

The Flexibility of Alternating dA—dT Sequences

Abstract

The flexibility of alternating poly (dA—dT) has been investigated by the technique of transient electric dichroism. Rotational relaxation times, which are very sensitive to changes in the end-to-end length of flexible polymers, are determined from the field free dichroism decay curves of four, well defined fragments of poly (dA—dT) ranging in size from 136 to 270 base pairs. Persistence lengths, calculated from the results of Hagerman and Zimm (Biopolymers (1981) 29, 1481–1502), are in the range 200–250 A. This makes alternating dA—dT sequences about twice as flexible as naturally occurring, “random” sequence DNA. Considering a bend around a nucleosome, for example, this difference in persistence length translates to an energy difference between poly (dA—dT) and random sequence DNA of 0. 17 kT/base pair or 1 kcal per 10 base pair stretch. This energy difference is sufficiently large to suggest that dA—dT sequences could serve as markers in DNA packaging, for example, at sites where DNA must tightly bend to accommodate structures.     

Recognition of Pyrimidine Dimers in DNA by the Incision Enzyme from <Emphasis Type="Italic">Micrococcus lysodeikticus</Emphasis>

A SIGNIFICANT proportion of the number of pyrimidine dimers induced in DNA by ultraviolet light is repaired by means of excision-resynthesis^1–3. Two enzymes that excise pyrimidine dimers from DNA have been purified from cells of a highly ultraviolet-resistant microorganism—Micrococcus lysodeikticus^4,5. One of these—an endonuclease—seems to recognize dimers and splits a phosphodiester bond near the dimers in DNA. The mechanism of recognition is not known; in particular, whether the incision enzyme recognizes either a local melting of DNA double helix or a specific chemical modification of one of DNA strands. If the former is correct, the incision enzyme should break the strand opposite to the dimer⁶ and the incision step in repair may lead to mutations⁶ or chromosome aberrations⁷.     

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.     

Studies on a mammalian cell protein (P8) with affinity for DNA in vitro

A protein (P8) found in cultured mammalian cells has been highly purified by DNA—cellulose chromatography alone. The protein is quite abundant, especially in human diploid fibroblasts, is readily extractable at low-salt concentration, and has an isoelectric pH close to neutrality. Its synthesis is not coupled to that of DNA, but it accounts for a greater proportion of the total soluble protein synthesis in growing than in resting cells. Among the proteins identified after DNA—cellulose chromatography, only P8 has affinity for single-stranded DNA but not for double-stranded DNA. It appears to have properties different from those of other DNA-binding proteins that have been described.     

Detection of DNA in Human Blastocyst Cavity Aspirate by Multiplex PCR

Preimplantation genetic testing (PGT) is a modern method of detection of chromosomal and genetic abnormalities in a human embryo before its transfer to the uterus. The genetic material is obtained by embryo biopsy. Here, we attempted to evaluate the efficiency of a method of noninvasive biopsy—aspiration of the blastocoel contents (blastocentesis) of human embryos. In this study, a biopsy was carried out human embryos with low morphological characteristics (3–4 CC according to Gardner grading) on day 6–7 of development. DNA obtained from the aspirate, as well as from the blastocyst, was analyzed by QF-PCR (quantitative fluorescent polymerase chain reaction) after whole genome amplification. In total, 24 blastocysts and aspirate samples obtained from them were analyzed; assayable DNA was found in seven (29%) aspirate samples from the blastocyst cavity, and this DNA was identical to blastocyst DNA in five (71%) cases. Thus, it was shown that, using aspiration of the blastocoel fluid of the human embryo, it is possible to obtain DNA suitable for analysis by molecular genetic methods. The features and advantages of the use of multiplex QF-PCR method combined with whole genome amplification for studying DNA obtained during aspiration of the blastocoel fluid are discussed. The prospects of DNA obtainment by the noninvasive biopsy method for preimplantation genetic testing (PGT) in the routine practice of infertility treatment and prevention of chromosomal and genetic abnormalities in newborns are considered.     

Genome editing system CRISPR/CAS9 and peculiarities of its application in monocots

Genome editing is a new methodology for DNA modification that has been developing in recent years. This review compares proposed methods of optimization and development of a modern genome editing system—CRISPR/Cas9—in monocots. Methodical approaches for in silico selecting target sites, designing an expression vector, transferring the vector expression cassette into plant cells, evaluating the results of the editing and nonspecific activity of the system, and obtaining modified plants free of foreign DNA are reviewed. The problem of legislative regulation and the prospects for using this method for commercial purposes are discussed.     

Ethoxyquin feeding to rats increases liver microsome-catalyzed formation of benzo(a)pyrene diol epoxide — DNA adduct

The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the animals had received ethoxyquin.     

Osmium ammine: Review of current applications to visualize DNA in electron microscopy

Summary— This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.     

Horizontal Gene Transfer: A Universal Phenomenon

According to the scientific literature, it is reasonable to consider that lateral transfer of genes is an usual mechanism of adaptation of the biological organisms to environmental stresses. Furthermore, from bacteria to cultured human cells, including fungi and plants, a large diversity of horizontal gene transfers—natural or artificial, experimental or deduced from sequence analysis—have been described. Therefore, the uncharacterized biodiversity—particularly in microbiology—associated with the universality of the horizontal gene transfer phenomenon leads to the consideration that dissemination of DNA from Genetically Modified Organisms (GMO) in biological environments, including food and soil, is uncontrolled and predictable.     

Markov analysis of viral DNA/RNA sequences

This work applies a previously published method for determining the order of a Markov chain to the DNA/RNA sequences of φX174, SV40 and MS2. In the first two cases rather long-range order is found—third and second order respectively—but zero order is the appropriate model for MS2. These results point to some inadequacies in previous informational calculations on virus genomes and lead to insights on features such as gene overlap and secondary structure.     

Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80 °C—a laborious process. A recent publication (Al‐Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H₂O₂, and so a common test of antioxidant status (resistance to strand breakage by H₂O₂) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H₂O₂. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H₂O₂ sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright © 2013 John Wiley & Sons, Ltd.