Flow dichroism of capsid DNA phages. I. Fast and slow T4B

Flow linear dichroic studies have been conducted on phage T4B in its fast and slow forms. The behavior of the phages is well represented by an equivalent ellipsoid model using the Peterlin-Stuart theory. The measurements permit the evaluation of the optical factor of the DNA in the phage and the rotary diffusion coefficient of the phage particle. Both these quantities change during the slow–fast conversion. The rotary diffusion results are in good agreement with those obtained by other workers with other methods. The optical factor is negative, indicating a net alignment of DNA helices parallel to the phage axis. The results exclude certain simplified models for the packaged DNA but do not lead to a unique structural conclusion. The flow dichroism experiment and its interpretation are described, and a simple method of calculating optical factors for complicated but cylindrically symmetric structures is presented.     

Interaction of dimeric intercalating dyes with single-stranded DNA.

The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine if TOTO exhibits any binding preference for dsDNA or ssDNA, transfer of TOTO from pre-labeled complexes to excess unlabeled DNA was assayed by gel electrophoresis. Transfer of TOTO from M13 ssDNA to unlabeled dsDNA proceeds to the same extent as that from M13 dsDNA to unlabeled dsDNA. A substantial amount of the dye is retained by both the M13 ssDNA and M13 dsDNA even when the competing dsDNA is present at a 600-fold weight excess; for both dsDNA and ssDNA, the pre-labeled complex retains approximately one TOTO per 30 bp (dsDNA) or bases (ssDNA). Rapid transfer of dye from both dsDNA and ssDNA complexes is seen at Na+ concentrations > 50 mM. Interestingly, at higher Na+ or Mg2+ concentrations, the M13 ssDNA-TOTO complex appears to be more stable to intrinsic dissociation (dissociation in the absence of competing DNA) than the complex between TOTO and M13 dsDNA. Similar results were obtained with the structurally unrelated dye ethidium homodimer. The dsDNA- and ssDNA-TOTO complexes were further examined by absorption, fluorescence and circular dichroism spectroscopy. The surprising conclusion is that polycationic dyes, such as TOTO and EthD, capable of bis-intercalation, interact with dsDNA and ssDNA with very similar high affinity.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

Studies of electron migration in DNA in aqueous solution using intercalating dyes.

The reactions of the hydrated electron (e-aq) and of the hydroxyl radical (OH) with double-stranded DNA in aqueous solution at room temperature have been studied through the use of the intercalating dyes, proflavine and ethidium. These dyes react with e-aq with rate constants of (2.5 +/- 0.2) - 10(10) M-1 - s-1 and (3.0 +/- 0.3) - 10(10) M-1 - s-1, respectively; the rate constant for the reaction of OH with proflavine is (1.0 +/- 0.2) - 10(10) M-1 - s-1. When these molecules are bound within the DNA structure both the yields and the rate constants of reaction with e-aq are reduced in a manner entirely consistent with a simple competition between the DNA bases and restricted dye molecules reacting with a bimolecular rate constant of about 2 - 10(9) M-1 - s-1. No evidence of free electron migration in the DNA was obtained, and an upper limit of five base pairs for the range of such migration was derived. Reactions of the hydroxyl radical with DNA-bound proflavine also lead to a rate constant of about 2 - 10(9) M-1 - s-1. These rate constants are in good agreement with rate predictions (per base unit) for a diffusion-controlled reaction with the DNA structure.     

Labelling of liposomes with intercalating perylene fluorescent dyes.

The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.     

Formation of free radicals and DNA breaks after pulsed laser irradiation of DNA complexes with intercalating dyes

Destruction of lambda phage DNA is studied under nanosecond pulse laser irradiation (lambda = 355 nm) of DNA-dye complexes in solution at 77K (dye--acridine orange, 8-methoxypsoralen, ethidium bromide). Free radicals induced by laser radiation are found to participate in DNA sugar-phosphate chain scission. It was observed that the quantity of DNA double-strand breaks correlated with that of the free radicals and that of oxygen influenced DNA laser destruction.     

Effect of intercalating dyes on the production of antibiotics by Micromonospora rosaria and Micromonospora purpurea.

The effect of treatment with various intercalating dyes on the ability to produce antibiotics in Micromonospora rosaria and Micromonospora purpurea was studied. Treatment with acriflavine resulted in a high frequency loss of antibiotic productivity in both species. In M. rosaria, the loss of antibiotic-producing ability appeared to be strain-dependent. In M. purpurea, up to 90% of colonies were found to have lost gentamicin-producing ability when protoplasts were used in the test. These antibiotic-nonproducing strains were further studied. The following observations were made: (1) Unlike the producing ability, the resistance to the antibiotics is a very stable character in both species. (2) Protoplast fusion analysis indicates that rosamicin-nonproducing characteristics of MR 217-AF2 and MR 217-AF3 strains induced by the acriflavine treatment is due to chromosomal mutation or rearrangement but not to loss of a plasmid. (3) Gentamicin-nonproducing strains of M. purpurea responded differently to the supplementation of streptamine or DOS in the culture medium. When supplemented with streptamine or DOS, some of these strains regained the ability to produce antibiotic, showing that the biosynthesis of intermediate was affected in these strains.     

Linear dichroism studies of nucleic acids. II. Calculation of reduced dichroism curves of A-and B-form DNA

We have calculated the uv linear dichroism for the A- and B-forms of DNA using π-π* transition moments and band components determined from the free DNA bases. The reduced dichroism (LD^R) as a function of wavelength is estimated is in the 220–300-nm region, for both the oriented-gas model and a simple exciton model. For B-form DNA, LD^R is obtained to ?1.48S (S being the orientation factor) over the whole wavelenth region by both models. For A-form DNA, LD^R is not constant, but changes monotonically from about ?1.15S at 220 nm to about ?1.35S to ?1.45S at 300 nm, depending on base combination and degree of interaction (?1.35S for the oriented gas). It is emphasized that a common assumption of a single “effective” transition moment of the principal band at 260 nm may not generally be made because of the extensive overlap of differently polarized bands. The possibility of using the reduced dichroism curve for characterizing the secondary structure of DNA is discussed.     

Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Flow dichroism of DNA: a new apparatus and further studies

A new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two-stranded DNA has been measured for the molecular weight range of 0.6 × 10⁶ to 125 × 10⁶, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec^?1 to 21000 sec^?1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro-dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm-Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.     

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.     

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Replication of M-13 DNA in plasmolysed Escherichia coli cells. Structure of a replicative intermediate with restricted binding of intercalating dyes.

DNA molecules with restricted binding of intercalating dyes are observed as replicative intermediates during the replication of bacteriophage M-13 duplex DNA in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Restriction of dye binding is abolished by heating the DNA to 80 degrees C, but can be recovered by slow cooling of the heat-treated DNA. Radioactive pulse-label incorporated by these molecules is found exclusively in elongated viral strands of more than one genome length. In the electron microscope this DNA fraction is seen to contain a significant number of duplex DNA rings with two single-stranded tails protruding from the same region of the ring. It is proposed that these structures arise by branch migration during the isolation of replicating molecules containing only one single-stranded tail. The topological constraint in these molecules is most likely caused by base-pairing between partially complementary regions of the two single-stranded tails.     

Quantitative flow dichroism. II. Form dichroism at ultraviolet wavelengths

Form dichroism has been predicted to make an important contribution to the dichroic spectra of rod-shaped macromolecules. Ultraviolet flow dichroism experiments on tobacco mosaic virus and bacteriophage fd in solvents of different refractive indices were unable to detect the phenomenon, indicating that for these viruses form dichroism is much less important than had been postulated.