Supramolecular organization of double-stranded DNA molecules in the columnar hexagonal liquid crystalline phase. An electron microscopic analysis using freeze-fracture methods

I present an electron microscopical analysis of the columnar hexagonal liquid crystalline phase of DNA. Freeze-fracture methods reveal that this phase is a lamellar structure, each layer (30 to 40 A thick) composed of DNA molecules aligned in parallel. Numerous defects can be seen in the structure, and their nature is determined. I show that they are mainly screw dislocations of both handedness. By this method it is possible to follow individual double-stranded DNA molecules in this highly packed structure. I show, moreover, that there is a local twist between DNA molecules along the screw dislocation lines and that this twist can be either right-handed or left-handed. The interest of such ultrastructural analysis is discussed in relation to the understanding of chromatin structure.     

Magnetic ordering of DNA liquid crystals

Sonicated calf thymus DNA with an average length of approximately 100 base pairs has been found to form a cholesteric liquid crystal at a concentration of approximately 250 mg of DNA/mL of solution. Immediately after preparation, small ordered domains of a few micrometers are formed, resulting in an opaque solution. This liquid crystal can readily be oriented in the magnetic field of an NMR magnet, resulting in a clear birefringent phase. The DNA molecules align with their helix axes perpendicular to the field so that the cholesteric pitch axis was parallel with the field. A pitch length of approximately 2.5 microns for the cholesteric phase was determined both from optical measurements (optical light rotation) and from NMR measurements (solvent diffusion). The observation that DNA molecules can be magnetically oriented opens up new possibilities for studying the structure and dynamics of the aligned DNA molecules.     

S-[2-(N7-guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane

The reaction of 1,2-dibromoethane and glutathione with DNA in the presence of glutathione S-transferase results in the formation of a single major DNA adduct, which can be released by thermal hydrolysis at neutral pH and separated by octadecylsilyl and propylamino high-performance liquid chromatography. The same DNA adduct is the only major one formed in livers of rats treated with 1,2-dibromo[1,2-14C]ethane. The DNA adduct was identified as S-[2-(N7-guanyl)ethyl]glutathione: (1) The chromatographic behavior was altered by treatment with gamma-glutamyl transpeptidase or Streptomyces griseus protease. (2) The molecular ions observed in positive and negative mode fast atom bombardment mass spectrometry were those expected for the structure when either glycerol or a mixture of dithiothreitol and dithioerythritol was used as the bombardment matrix. (3) The two-dimensional 1H NMR correlated spectroscopy spectrum of the DNA adduct was compared to the spectra of glutathione, oxidized glutathione, and N7-methylguanine and found to be consistent with the assigned structure. No evidence for in vitro or in vivo opening of the guanyl imidazole ring was observed under these conditions. The structure of the adduct supports a pathway involving enzyme-catalyzed conjugation of 1,2-dibromoethane with glutathione, non-enzymatic dehydrohalogenation of the resulting half-mustard to form a cyclic episulfonium ion, and attack of the N7 nitrogen of DNA guanine on the episulfonium ion to generate this major DNA adduct, which may be related to the carcinogenicity of this chemical.     

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.     

Probing DNA quaternary ordering with circular dichroism spectroscopy: Studies of equine sperm chromosomal fibers

Studies are described which strongly support a cholesteric liquid crystal-like quaternary structure for the DNA molecules of a biologically native chromosomal preparation from equine sperm cells. Discrete chromosomal fibers released from the head pieces of equine spermatozoon cells were prepared intact and probed for liquid crystalline ordering using reflectance and linear dichroism spectroscopy. Assuming cholesteric liquid crystalline order for the DNA molecules within the chromatin fibers, parameters measured experimentally were used to calculate the circular dichroism (CD) of the fibers. The calculated results compare remarkably well with the experimentally measured CD of the sperm chromosomal fibers and suggest a specific cholesteric liquid crystal-like quanternary structural ordering of DNA molecules in equine sperm chromatin fibers. The potential of CD spectroscopy as a tool for the study of long-range ordering of macromolecules is discussed.     

Theoretical analysis of DNA branch migration in the presence of a slow reversible initiation step

Branched DNA structures include several DNA regions connected by three- or four-way DNA junctions. Branched DNAs can be intermediates in DNA replication and recombination in living organisms and in sequence-specific DNA targeting in vitro. Branched DNA structures are usually metastable and irreversibly dissociate to non-branched products via a DNA strand exchange process commonly known as DNA branch migration. The key parameter in the DNA dissociation process is its characteristic time, which depends on the length of the dissociating DNA structure. Here, we predict that the presence of a slow reversible initiation step, which precedes DNA branch migration, can alter, to almost linear dependence, the "classic" quadratic dependence of the dissociation time on the length of the dissociating DNA structure. This prediction can be applied to dissociation of Y-like DNA structures and double D-loop DNA hybrids, which are DNA structures similar to replication bubbles. In addition, the slow initiation step can increase the effect of DNA sequence heterologies within the structure on its kinetic stability. Applications of our analysis for genetic manipulations with branched DNA structures are discussed.     

Supramolecular ordering of DNA in the cholesteric liquid crystalline phase: an ultrastructural study.

Aqueous solutions of 146-base pair DNA fragments form a cholesteric liquid crystalline phase in the range of about 160-290 mg/ml. We present a structural analysis of this phase by comparing the data obtained from polarizing and electron microscopy. This phase shows multiple aspects or "textures" which are presented and interpreted. They mainly depend on the orientation of the structure relative to the observation plane and on the nature, distribution, and amount of defects present in the phase. These defects are then analyzed with the two methods, and the molecular orientations can be defined precisely in their core. The biological interest of such structural analyses is discussed in relation with the understanding of chromatin structure and function.     

Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs

Covalent modification of DNA by antineoplastic agents represents a potent biochemical lesion which can play a major role in drug mechanism of action. The ability to measure levels of DNA covalent modifications in target cells in vivo may, therefore, be seen as the ultimate form of therapeutic drug monitoring. Additionally, elucidation of the structure of critical DNA adducts and definition of their role in tumour cell cytotoxicity will provide more selective targets for rational drug design of new cancer chemotherapeutic agents. High-performance liquid chromatography has contributed significantly to all these areas. In vivo levels of nucleic acid covalent modifications are in the range of 1 in 10⁵–10⁸ nucleotides precluding the use of conventional high-performance liquid chromatographic detection methods. Several classes of natural product anticancer drugs have been shown to bond covalently to nucleic acids under optimal laboratory conditions. These have proved more accessible to high-performance liquid chromatographic analysis because of their lipophilicity and strong UV chromophores. However, the majority of experimental evidence to date suggests that with the exception of mitomycin C and morpholino-anthracyclines these compounds do not exert their primary mechanism of action through nucleic acid covalent modification. DNA adducts of alkylating and platinating agents are more difficult to detect by high-performance liquid chromatography and can be chemically unstable. These compounds interact with DNA on the basis of chemical kinetics. Thus, the principle sites of attachment tend to be with the most nucleophilic base (guanine) at its most reactive centre (N-7 position). Limited in vivo high-performance liquid chromatographic studies with all classes of anticancer drugs indicate a much more complex pattern of adductation than would have been anticipated from in vitro studies alone. Some of these differences are probably due to methodological artefacts but these studies stress the need for sensitive detection methods and reliable sample preparation (nucleic acid extraction and digestion techniques) when attempting to determine nucleic acid covalent modifications by anticancer drugs.     

"External chromophores" and intensive bands in circular dichroism spectra of liquid-crystalline microphases of nucleic acids

From the analysis of CD-spectra of liquid crystalline microphases formed from DNA molecules in complexes with "external chromophores" requirements are formulated for the compounds to be used in revealing peculiarities of spatial structure of nucleic acids liquid crystalline microphases. These requirements satisfied it is possible to record the CD-spectra having intensive bands in the regions of chromophores absorption. These bands prove the helical pattern of the spatial structure of nucleic acid liquid crystalline microphase.     

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.     

氩离子注入介导麻黄基因组DNA转化获得产麻黄碱重组酵母菌

通过氩离子(Ar )注入介导蓝麻黄基因组DNA在异常汉逊酵母(Hansenula anomala)中随机转化,转化后的酵母菌经BTB指示性辅助筛选、斜面传代、液体培养、铜铬盐定性检识和RP-HPLC定量检测,获得了遗传稳定的以葡萄糖为碳源、NaNO3为氮源生物合成麻黄碱和(或)伪麻黄碱的重组酵母菌3株。液体培养72h,RP-HPLC测试胞外麻黄碱和伪麻黄碱的最高产量分别为11.87mg/L和4.11mg/L;胞内伪麻黄碱最高含量为294.86mg/g干细胞,胞内麻黄碱未检出。分析了Ar 注入介导蓝麻黄基因组DNA在酵母菌中的遗传转化效率,探讨了麻黄基因组DNA大分子的完整性对其在酵母菌中遗传转化的影响。     

New observations on the twisted arrangement of Dinoflagellate chromosomes

The Dinoflagellate Prorocentrum micans has been studied in classical and high voltage transmission electron microscopy, with the help of a goniometric stage. The general structure of the nucleus is analysed with special reference to the links observed between chromosomes and the nuclear envelope, the nucleoplasm and the nucleolus. The chromosomes present stacked series of nested arcs which are studied in detail. The sense of the arcs can be changed by a simple tilt of the section. These arcs do not correspond to DNA filaments with a genuine bend but to an illusion created by the overlap of layers of filaments whose orientation turns along the chromosome axis. — The transversal orientation of DNA and the examination of defects allow to rule out the polytenic hypothesis. It is clear that this hypothesis does not apply to bacterial nucleoids, which however can form series of nested arcs as in Dinoflagellate chromosomes. — The twisted arrangement of Dinoflagellate chromosomes is that of a liquid crystal of the cholesteric type. DNA is known to self assemble into cholesteric phases and this affords informations on the origin of the elongated shape of chromosomes and on the mechanisms of condensation and aggregation observed in this particular chromatin.     

DNA in human and stallion spermatozoa forms local hexagonal packing with twist and many defects

In human and other mammal sperm nuclei, DNA is packed in a highly condensed state, the structure of which remains unsolved. Cryoelectron microscopy of vitrified sections provides a first direct view of the local arrangement of the nucleoprotamine filament. DNA aligns in parallel in layers and its orientation rotates along a single-twist direction as in a cholesteric liquid crystal. The structure contains numerous defects, which introduce locally double-twist configurations. Destruction of the SS bonds with dithiotrehitol relaxes the twist and favors the extension of the hexagonal close packing of the filaments, though keeping constant their interfilament distance.     

Asymmetric Transcription of Bacteriophage Mu-1

The deoxyribonucleic acid (DNA) of bacteriophage Mu-1 can be separated into its complementary strands by poly(U,G) binding and equilibrium centrifugation. DNA-ribonucleic acid (RNA) hybridizations in liquid show that more than 98% of "early" phage-specific RNA and over 96% of "late" messenger species bind to the heavy [poly(U,G)-binding] strand of Mu-1 DNA. A small (1.45%) but significant amount of late RNA binds to the light strand. The significance of this RNA fraction is discussed in connection with the peculiar structure of denatured and reannealed Mu-1 DNA.     

Low-energy electrons inside active DNA models: A tool to elucidate the radiation action mechanisms

To postulate radiation action mechanisms and to test them by Monte Carlo simulation, a complex computer model was developed consisting of major components for the generation of a radiation spectrum, biomolecular structures, and electron track structures in liquid water. As the radiation source¹²⁵I is employed here; it is an excellent test radiation due to its exactly localized position in the DNA molecule and high biological toxicity as a consequence of the emission of short-ranging Auger electrons. A linear DNA plasmid model (Pomplun 1991) which can actively respond to radical attack (Terrissol and Pomplun 1994) has been modified into a nucleosome model representing the double-helix of DNA with 146 basepairs and more than 9000 atoms surrounding the histones. The introduction of this new target structure allows a more realistic simulation of cellular conditions. Using the model's decay accumulation aspect, the situation of many break and survival experiments can be approximated and the influence of several cellular parameters tested. As a first step, a correlation between the size of energy depositions and strand-break patterns was sought.     

Direct DNA extraction for PCR-mediated assays of soil organisms.

By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.     

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.

A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.     

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time (t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.     

Early Intracellular Events in the Replication of Bacteriophage T4 Deoxyribonucleic Acid: V. Further Studies on the T4 Protein-Deoxyribonucleic Acid Complex 1

Soon after infection parental deoxyribonucleic acid (DNA) enters a structure sedimenting fast to the bottom of a sucrose gradient. The addition of chloramphenicol (CM) prevents formation of this structure, whereas treatment with Pronase releases DNA which sediments thereafter with the speed characteristic of phenol-extracted replicative DNA. It is assumed therefore that the structure responsible for fast sedimentation of replicative DNA is a newly synthesized protein. Those fast-sedimenting complexes contain preferentially the replicative form of parental DNA; this was proven by density labeling experiments. Progeny DNA labeled with (3)H-thymidine added after infection can also be detected preferentially in this fast-sedimenting moiety. The association of the DNA with the complexing protein is of a colinear or quasi-colinear type. This was proven by introducing double-strand scissions into DNA embedded in the replicative complex; double-strand scissions do not liberate DNA from the fast-sedimenting complex. Despite the apparent intimate relation between protein and DNA, DNA residing in complexes is fully sensitive to the action of nucleases. Shortly prior to the appearance of the fast-sedimenting complex, parental DNA displays still another characteristic: at about 3 min after infection, it sediments faster than reference, but sizeably slower than the complex which appears at roughly 4 to 5 min after infection. The transition between these two fast-sedimenting types of moieties is not continuous. This fast-sedimenting intermediate, which appears at 3 min after infection, cannot be inhibited by the addition of CM either at the moment or prior to infection. Fast-sedimenting intermediate can be destroyed by sodium dodecyl sulfate, Pronase, or phenol extraction. The progeny DNA labeled with (3)H-thymidine between 3 and 3.5 min after infection can be recovered in fast-sedimenting intermediate. The contribution of newly synthesized progeny DNA is so small that it cannot be detected as a shift of the parental density in a density labeling experiment. Small fragments of progeny DNA recovered in fast-sedimenting intermediate are not covalentlv attached to parental molecules and represent both strands of T4 DNA.