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Preparation and properties of native crab d'AT

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Chapter
Mercury is distributed widely in the earth’s crust, in sea, ground and rain water. Importantly, all phyla and species naturally contain traces, present either as inorganic or organometallic compounds, or both.1,2 A biological role for the element is thus far undefined; however, it is present in rat liver chromatin3 and methyl mercury induces hepatic protein synthesis in this species.4 It has been known that mercury enters into biological life cycles; however, the awareness that inorganic mercury can be converted into organometallic compounds by bacteria and higher organisms has recently stimulated further interest both in the chemistry and toxicological potential of this element.5–9 This biological conversion of inorganic mercury into organic mercury is particularly significant since extensive industrial and agricultural usage of mercurials affects and increases its distribution in specific regions. Moreover, the burning of fossil fuels generates environmental mercury in amounts comparable to those from industrial processes.10
1. DNA ligase isolated from plants catalyzes the formation of covalently linked circular conformations of plant and animal DNA's. Such circular structures are formed only in the presence, but not in the absence of specific plant and animal hormones. Hormones have no cyclization effects on prokaryotic DNA.2. The above DNA circles consist, to a larger extent, of covalently joined internal loops and not end-to-end links as in γ-phage DNA.3. The capability of eukaryotic DNA to form internal loops in response to specific hormones decreases with advancing age.
Article
The equilibrium and the stoichiometry for the reversible complexing of silver ion by DNA have been studied by potentiometric titrations, proton release pH-stat titrations, and by spectrophotometry. The complexing reactions involve primarily the purine and pyrimidine residues, not the phosphate groups. There are at least three types of binding (types I, II, and III), of which the first two have been intensively studied in this work. The sum of type I and type II binding saturates at one silver atom per two nucleotide residues. In the type I and type II reactions, zero and one proton, respectively, are displaced per silver ion bound. At pH 5.6, the reactions occur stepwise, type I being first, while at pH 8.0, they occur simultaneously. The silver ion binding curve is very sharp at pH 8, indicating a cooperative reaction. The strength of the binding increases with increasing GC content. Type I binding is more important for GC-rich DNA's than for GC-poor ones. Denatured DNA binds more strongly than does native DNA. The silver ion complexing reaction is chemically and biologically reversible. We propose that type II binding essentially involves the conversion of an hydrogen bond of a complementary base pair to an N—Ag—N bond. The nature of type I binding is less clear, but it may involve a π interaction with stacked bases. The buoyant density (ρ0) of DNA in a Cs2SO4 density gradient increases when the DNA reacts with silver ion. The buoyant density change is about 0.15 g./ml. for 0.5 silver bound per nucleotide. The large buoyant density changes and the selective nature of the complexing reaction make it possible to perform good separations between native and denatured DNA or between GC-rich and GC-poor native DNA's by density gradient centrifugation.
Article
The one-dimensional Ising model, with nearest neighbor correlation only, suitably modified, is used to explain the observed linear dependence of melting temperature of copolymeric DNA with GC content. Transition curves are plotted for regular, random, and Markoff distribution of base pairs for various values of a correlation parameter U between nearest neighbor bonds. Exact analytic formulas are given for fraction of bonds intact at a particular temperature for various regular distributions for all U and approximate ones for random and Markoff distributions for small U. A scheme is indicated for further improvement. The model, in principle, makes it possible to estimate the statistical distribution of base pairs from the detailed shape of the transition curve.
Article
SYNOPSIS. Data concerning the DNA and RNA content of 3 Trypanosoma species (T. equiperdum, T. gambiense and T. cruzi) are given. Kinetoplastic DNA was fractionated and separated from nuclear DNA by ultracentrifugation in a CS2SO4 gradient after complexation by mercury or silver ions. Buoyant densities in the analytic ultracentrifuge, base composition and melting point of these DNA's were studied.
Article
Metal $ions^{1..10}$ are known to bind to DNA. A number of metal ions have been studied and their binding established. $Cu(II)^{8,9,10}$ and $Ag(I)^4$ have been widely studied and ample literature exists on these two metals. On the other hand, the interaction of DNA with gold(III) (which does not occur as $Au^{3+}$ in solution, but rather exists in square planar $complexes^{11}$) has seldom been considered. During the course of our work on this phenomenon Gibson and $co-workers^{12}$ have published a paper recently reporting the results on binding of gold(III) with adenine nucleotides.
Article
Chromatin structure can be probed by cross-linking DNA in situ using trioxsalen and irradiation with UV light. Presumably DNA within a nucleosome is protected from cross-linking so that this region appears as a single-strand loop in the electron microscope under a condition in which single-strands and double-strands are distinguished. Unprotected regions appear as duplex due to cross-linking. We have used this approach to investigate the structure of chromatins containing satellite DNAs of Drosophila nasutoides. We have previously shown that D. nasutoides has an unusually large autosome pair which is almost entirely heterochromatic. Its nuclear DNA reveals four major satellite components amounting up to 60% of the total genome. All of them are localized in this large heterochromatic chromosome. We wish to ask whether chromatins containing different satellite sequences have different arrangements of nucleosomes. Our results from cross-linking experiments show that all DNA components including main band DNA have different patterns of protected and unprotected regions: (a) The length distributions of protected regions show multiple peaks with the smallest unit lengths being 200 nucleotides for main band DNA, 180 for satellites I, II and III, and 160 for satellite IV. (b) The amounts of unprotected regions, presumably internucleosome DNA, vary from 16% for main band DNA to 60% for satellite IV, suggesting that satellite chromatins have fewer nucleosomes per given length of chromatin than main band DNA chromatin. The spacings between nucleosomes appear to be random in satellite chromatins.
Article
CV1 and A549 cells were grown in the presence of64Cu porphyric complex,64CuCl2, or67CuCl2. Radioactive copper determinations were performed on whole cells and on isolated cellular DNA.125IUdR was used to calibrate the particular extraction and purification procedures we developed because of the half-lives of64Cu and67Cu. The results obtained have shown that some radioactive copper atoms remained firmly bound to the DNA molecule. Their amount was of the same order when using two different DNA isolation methods for the two cell lines studied. No significant differences were found when64Cu was used as CuCl2 or as porphyric complex.
An explanation is sought for the observation that only 60 % of the GC base pairs of crab poly d(A-T) · poly d(T-A) are strong binding sites for actinomycin D.The size distribution was measured for the oligonucleotides obtained by ribonuclease T1 digestion of the RNA transcription product from crab poly d(A-T) · poly d(T-A). This distribution shows that the G residues are randomly distributed along each strand.The association constant and the number of base pairs per binding site for the binding of AMD by poly d(T-G) · poly d(A-C) is measured as about the same as for a natural DNA. Therefore, G does not require a neighboring purine to be a binding site.Wells22 and Wells and Larson7 have recently shown that poly d(T-G-A) · poly d(A-C-T) does not bind actinomycin D. We conclude that a combination of steric interactions for randomly distributed GC base pairs and the nonbinding character of the TGA sequence is probably responsible for the binding data for crab poly d(A-T) · poly d(T-A).
Article
A new procedure for the preparation of yeast mitochondrial DNA has been developed. Its main advantages over previous methods are its rapidity, its flexibility in scale, and the quantitative yield in mitochondrial DNA.Several properties of the mitochondrial DNA have been investigated. It has been found that yeast mitochondrial DNA (18% G + C) has a higher buoyant density, in the presence of Ag+, than Haemophilus influenzae (38% G + C), or yeast nuclear DNA (39% G + C). Both the abnormally high buoyant density in Cs2SO4Ag+ density gradients and the abnormally high elution molarity from hydroxyapatite columns appear to be due mainly to the A + T-rich stretches present in mitochondrial DNA.G + C-rich “molecules” (22% G + C; 13 s) have been isolated from the bulk of mitochondrial DNA by virtue of their lower elution molarity from hydroxyapatite, thus providing the first evidence that yeast mitochondrial DNA has an “intermolecular” compositional heterogeneity at a size level of about 1.5 × 106 daltons. Fragments having a G + C content of 24% and an S20,w = 6.6 s could be prepared from these molecules by degradation with spleen acid DNase and centrifugation in a Cs2SO4Ag+ density-gradient.
Article
A relatively simple and rapid method has been developed which allows quantitation of the rate of production of single-strand scissions in polydisperse populations of DNA molecules. This method, which is based on determination of the progressive loss of the rapid renaturability (zippering-up) of cross-linked DNA, was applied as a quantitative assay of strand breakage by ionizing radiation and by DNase I. Radiosensitivity to single-strand scissions was proportional to the molecular size but independent of the average base composition or substitution of bromouracil for thymine. The protective effects of cysteine and cysteamine, and self-protection at higher DNA concentrations were demonstrated.
1.1. The effects of alteration of pH and of addition of dimethylsulfoxide on the buoyant behavior of single- and double-stranded RNA's and DNA's in Cs2SO4 have been investigated. Conditions have been found in which single-stranded RNA neither precipitates nor adsorbs to the plastic centrifuge tubes used in preparative ultracentrifuges.2.2. The redistribution of Cs2SO4, dimethylsulfoxide, and water at equilibrium in 3-ml columns of solution in the ultracentrifuge has been determined experimentally. The density distributions have also been determined. The addition of 10 vol.% dimethylsulfoxide to the initial solution approximately doubles the density gradient at the isodensity position in the cell. The density gradients measured in preparative ultracentrifuge experiments were used to calculate apparent buoyant densities from the results obtained in the analytical ultracentrifuge.3.3. The tertiary buoyant solvent, Cs2SO4, 10 vol.% dimethylsulfoxide, and water, can be used to separate duplex DNA, single-stranded RNA, and double-stranded RNA.
Article
The mitochondrial DNA's from two “grande” yeast strains and three cytoplasmic “petite” mutants were isolated by hydroxyapatite chromatography and investigated in their chemical and physical properties.The buoyant densities of all DNA's were equal to 1.683 g/cm3, except for one from a petite strain which had a density equal to 1.678 g/cm3. The mitochondrial DNA's from the petite mutants were “reversible”, their buoyant densities and their ultraviolet absorptions showing very little or no increase after heating and fast cooling. In contrast, the DNA's from the grande cells showed an increase in buoyant density of 0.010 to 0.011 g/cm3 and a residual hyperchromicity of 10 to 12% upon the same treatment. In this case, both density and residual hyperchromicity were lowered by thermal treatment at 65 °C.The guanine plus cytosine contents of the DNA's from the grande strains were found to be 17.4 and 16.8%. Those of the DNA's from the petite mutants were 15.5, 15.6 and 12.6%. For all mitochondrial DNA's, the base compositions calculated from their buoyant densities and Tm values did not fit these obtained by analysis.A striking compositional heterogeneity was indicated by the ultraviolet melting curves of all mitochondrial DNA's. The distribution of the melting components of the DNA's from the grande strains was remarkably different from that of the DNA's from the petite strains. The first one was broad and asymmetric, the second one was multimodal with a small number of components. Furthermore, the DNA's from the different petite mutants showed distinct melting patterns.
Article
The changes in absorption spectra in the visible region observed on adding different naturally occurring and synthetic DNA duplexes to solutions of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein indicate that the mercurial reacts with polynucleotides of this type. The reaction is reversible as proved by adding excess of KCN which restores the original spectra of the free dye. The interaction is characterised also by quenching of the fluorescence of the dye and the induction of optical activity in it. The extent of these spectral effects depends strongly on the (A+T) content of the complexed DNA and decreased in the order: poly [d(A-T)], Clostridium perfringens DAN, Escherichia coli DNA, Micrococcus luteus DNA and poly(dC). From equilibrium-dialysis experiments the same order in affinity is obtained when these poly-nucleotides are at equilibrium with the same concentration of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein. From the changes produced by different mercurials in the ORD spectra and viscosity of a DNA solution it has been concluded that 4,5-dibromo-2,7-di(acetatomercuri)-fluorescein does not cause any drastic alteration of the secondary structure of DNA.
Article
Yeast DNA has been fractionated in preparative Ag+-C-S2SO4 density gradients. After complexing with silver ions at a pH of about 7, the rDNA appears in a defined heavy satellite component in the gradient. For our particular strain, the satellite represents about 15% of the total nuclear DNA and has been identified as the gamma-DNA. In alkaline CsCl density gradients, the satellite DNA forms two bands, of which the light component hybridizes with ribosomal RNA.
Article
The enthalpy deltaH, entropy deltaS, and the temperature Tm of the conformational transition of poly[d (A-T)] from the ordered to the randomly oriented state have been determined at pH 6.8 with the help of an adiabatic differential scanning calorimeter in Na2SO4 solutions of increasing ionic strength. Spectrophotometric denaturation experiments supplemented the calorimetric measurements. All thermodynamic parameters were found to vary strongly with salt concentration: both deltaH and Tm increase linearly with the logarithm of the mean molal activity alpha plus or minus of Na2SO4. However, whereas the dependence of Tm on salt activity remains linear over the entire salt concentration range employed deltaH decreases abruptly in the most concentrated Na2SO4 solutions. The entropy of melting changes with salt concentration in a pattern similar to that displayed by deltaH. The data on deltaH as well as data derived from the maximum slopes of the calorimetric heat denaturation curves were used to calculate the cooperative length Lh, the stacking free energy epsilon, and the cooperativity parameter sigma of poly[d(A-T)] as a function of ionic strength. Lh decreases with increasing salt concentration whereas sigma increases. Epsilon assumes more positive values with increasing salt molality. These changes then are in agreement with the generally held belief that an increase in salt concentration leads to an increase in the "loop" content of the copolymer.
Article
Crab (dA-dT)n was isolated from the testes of Cancer borealis by a procedure involving separation of DNA and segregation of the satellite fraction by Hg2+ binding/Cs2SO4 density gradient ultracentrifugation. The titration of crab (dA-dT)n samples at 10 degrees indicated a sharp absorbance change at pH 11.98 in agreement with the pHm value observed for synthetic poly(dA-dT) under identical conditions. The reversal of the titration, however, resulted only in about 50% recovery of the original absorbance (at 260 nm) in marked contrast to the complete reversibility of the synthetic material. pH-jump experiments were carried out for the purpose of characterizing the rates and mechanisms of conformational transitions brought about by changes in the solution environment. It was found that the disintegration of the putative native structure of crab (dA-dT)n starts with a very fast reaction (occurring within the 6-msec deadtime of the instrument and comprising 65% of the total absorbance change) and it is completed via a slower first-order reaction (k = 66 sec minus 1). It is postulated that the first process is due to the rapid untwisting of end regions and, perhaps, some short hairpin-like helical branches present on the macromolecules. The second reaction is believed to be the end-to-end type unwinding of the double-helical backbone of crab (dA-dT)n. In the presence of low concentration (3 mug/ml) of Hg2+ ions the overall rate of disintegration process decreased drastically. pH jumps from pH values above pHm to values below were used to study the rates of absorbance changes corresponding to the refolding of the strands of denatured crab (DA-dT)n. A concentration independent process consisting of two phases was observed. The first phase was a gradual nonexponential process spanning the first second of the reaction, and the other, a very slow first-order process characterized by the rate constant value of 0.053 sec minus 1. It is proposed that the first part of the process (involving about 24% of nucleotide residues) is an intramolecular formation of helical hairpins (frequently interrupted by mismatching bases) and the second part is a manifestation of some association of the extant unpaired bases during the folding of the branched structure. Refolded crab (dA-dT)n samples when subjected again to pH greater than pHm in the stopped-flow apparatus displayed not the disintegration pattern of the native crab (dA-dT)n but rather that of synthetic poly(dA-dT. The marked facility of crab (dA-dT)n macromolecules for rapid conformational transitions induced by slight changes in the solution environment might be relevant to the biological function of this DNA.
Article
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Bacteriophage T5-induced DNA polymerase shows an initial phase of rapid synthesis, followed by a slower steady rate for much longer periods, with short DNA primer-templates (400 to 600 nucleotides long), in vitro. On extrapolating the line of steady rate back to 0 min, an intercept is obtained on the ordinate. With large DNA primer-templates, such as denatured T5 DNA (average chain length approximately 50,000 bases), the rate of synthesis remains constant and is equal to the initial rate obtained with short primer-templates. The zero time intercept was proportional to the amount of enzyme used and independent of temperature. Polymer challenge experiments indicate that the initial phase of rapid synthesis can be attributed to the processive mode of synthesis by T5 DNA polymerase. After synthesizing a stretch of DNA processively for about 200 nucleotide residues, the enzyme apparently forms a "dead-end complex" with the primer-templates used and must dissociate from the primer-template in order to resume synthesis. The average size of the product made processively, during various phase of synthesis, remains invariant and is in good agreement with the size of the zero time intercept per enzyme molecule.
Article
The nature of interaction of palladium (II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.
Article
The nature of interaction of Au(III) with nucleic acids was studied by using methods such as uv and ir spectrophotometry, viscometry, pH titrations, and melting-temperature measurements. Au(III) is found to interact slowly with nucleic acids over a period of several hours. The uv spectra of native calf-thymus DNA 9pH 5.6 acetate buffer containing (0.01M NaCIO4) showed a shift in λ max to high wavelengths and an increase in optical density at 260 nm. There was a fourfold decrease in viscosity (expressed as ηsp/c). The reaction was faster at pH 4.0 and also with denatured DNA (pH 5.6) and whole yeast RNA (pH 5.6). The order of preference of Au(III) (as deduced from the time of completion of reaction) for the nucleic acids in RNA > denatured DNA > DNA. The reaction was found to be completely reversible with respect KCN. Infrared spectra of DNA-Au(III) complexes showed binding to both the phosphate and bases of DNA. The same conclusions were also arrived at by melting-temperature studies of Au(III)-DNA system. pH titrations showed liberation of two hydroxylions at r = 0.12 [r = moles of HAuCl4 added per mole of DNA-(P)] and one hydrogen ion at r = 0.5. The probable binding sites could be N(1)/N(7) of adenine, N(7) and/or C(6)O of guanine, N(3) of cytosine and N(3) of thymine.
Article
Biochemistry. 1982 Jan 5;21(1):62-6 The binding of Hg(II) to poly(dA-dT) has been examined with proton NMR spectroscopy. Addition of HgCl2 between r (Hg2+/nucleotide) = 0 and 0.25 results in loss of the exchangeable imino N3H resonance of thymine, indicating preferential binding at this site. The nonexchangeable base resonances AH8, AH2, and TH6 shift their intensity downfield in a cooperative manner, indicating complexation which is slow on the NMR time scale and changes in the polymer conformation upon binding. At r = 0.25, the polymer is cross-linked, and an increase in temperature does not result in denaturation of the polymer, as evidenced by the thymine proton resonance chemical shifts. The chemical shifts of the AH2 and T(CH3)5 base resonances allow some general conclusions to be made about the stereochemistry of this complex.
Chapter
Summary This document is part of Subvolume C ‘Spectroscopic and Kinetic Data. Physical Data I’ of Volume 1 ‘Nucleic Acids’ of Landolt-Börnstein - Group VII Biophysics.
Article
In vitro transcription of native DNA isolated from mature bacteriophage λ was studied using highly purified preparations of DNA-dependent RNA polymerase isolated from E. coli W. Half-length segments of sheared λ DNA were separated by density-gradient centrifugation, and the RNA polymerase products synthesized on the whole λ DNA template and on each of its separated halves were characterized with regard to their nearest-neighbor nucleotide frequencies, base composition, average chain-length, sedimentation velocity, and ability to anneal with specific segments of the template. The priming efficiencies of the λ DNA halves were compared, and the influence of certain alterations in the secondary or tertiary structure of the λ DNA template on the RNA products formed in vitro was examined.
Chapter
Summary This document is part of Subvolume C ‘Spectroscopic and Kinetic Data. Physical Data I’ of Volume 1 ‘Nucleic Acids’ of Landolt-Börnstein - Group VII Biophysics.
Article
A basic aim of chemotherapy is clearly to obtain a selective effect between a normal host cell and the invading cell, be it bacterial, protozoal, or cancerous. This selectivity may be achieved by differences in distribution, comparative biochemistry, or comparative cytology [1], the damage induced eventually rendering the attacked cell more susceptible to the host’s defense mechanisms.
Chapter
This chapter describes methods for the isolation of deoxyribonucleate, with alternating deoxyadenylate and thymidylate residues from crabs. A deoxyribonucleate with physical characteristics similar to those of a copolymer of alternating deoxyadenylate and thymidylate residues is found as a minor component of the deoxyribonucleate of a number of species of crab. This interesting substance is characteristic of families in the crustacean superfamily Brachyrhyncha. Crab deoxyadenylate-thymidylate copolymer is present in some species as about 10% and in others as about 30% of the total deoxyribonucleate in testicular tissue, and in analogous proportions in other tissues. Two methods are described for isolating this relatively simple deoxyribonucleate free of the major crab deoxyribonucleate, which has about 40 mole percent of guanine-cytosine base pairs. The first depends on the ready ability of crab thymidylate-deoxyadenylate copolymer to re-form a double-stranded structure after heat denaturation. In this case, renatured nucleate is freed from the denatured main component on a methylated albumin kieselguhr column. The second procedure depends on the ability of mercuric ion to bind preferentially to crab deoxyadenylate- thymidylate copolymer. The two types of crab deoxyribonucleate are separated by density gradient centrifugation in aqueous cesium sulfate.
Article
Fishes were exposed to graded series of concentrations of cadmium chloride for acute toxicity studies. The MAC value deduced was 1.25 mg/L of cadmium chloride in 50 litres of water for 30 days. A safety concentration of 1.20 mg/L of Cadmium chloride 1-50 was selected for this study. Exposed fishes appeared lethargic when compared to the control fish. Exposed fishes showed erratic movements, loss of equilibrium, gradual onset of inactivity etc.when compared to the control fish. The brain, liver, muscle, kidney and gill of the exposed fish accumulated 0.53, 22.34, 0.063, 28.9 and 0.59 mg of Cadmium/gm samples after 28d of exposure, respectively. Around 18.62, 61.25, 65.86, 40.11 and 76.1% excretion / removal of cadmium was marked afetr 28d of recovery. Significant depletion of total ATPase activity in the cadmium chloride exposed fish tissues, when compared to control fish tissues was marked. The exposed fish muscle tissue was highly affected, where 76.9% depletion in the activity was noted. The exposed brain tissue was least affected. This depletion in the enzyme activity affected the movements of ions across the membrane and severely affected the energy metabolism and disturbed energy budget of the fish.
Chapter
This chapter discusses the use of cesium sulfate for equilibrium density gradient centrifugation. Equilibrium density gradient centrifugation is one of the most useful tools for fractionation and characterization of DNA. The two cesium salts most commonly employed in this procedure are CsC1 and Cs2SO4. The properties of density gradients prepared with these salts are rather different. At similar rotor speeds, Cs2SO4, which forms approximately twice as steep a gradient as CsCl, is preferable for fractionation of DNA's with widely different buoyant densities, for example, dAT versus dABU or normal unsubstituted versus bromoor iodouracil-labeled DNA. On the other hand, CsCl is better suited for routine determination of the guanine + cytosine (G + C) content of DNA, because in this solvent, there is an almost linear relationship between the percentage of G + C (20–80%) and the buoyant density of DNA. In Cs2SO4, there is a much less and nonlinear dependence of density on G + C content. DNA is much more heavily hydrated in the Cs2SO4gradient, with the density averaging 1.4 g/cm3 as compared with 1.7 g/cm3 in CsCl. The chapter also explains that RNA can be banded in the Cs2SO4 gradient at a concentration of about 1.6 g/cm and discusses the other specific advantages of Cs2SO4 over CsC1.
Yeast DNA has been fractionated in preparative Ag+-Cs2 SO4 density gradients. After complexing with silver ions at a pH of about 7, the rDNA appears in a defined heavy satellite component in the gradient. For our particular strain, the satellite represents about 15% of the total nuclear DNA and has been identified as the γ-DNA. In alkaline CsCl density gradients, the satellite DNA forms two bands, of which the light component hybridizes with ribosomal RNA.
Article
Additional proof has been obtained for the fact that the triplet state in DNA and poly dAT resides at the thymine residue by comparing the intensities of the triplet ESR and phosphorescence signals from DNA's from various sources having different (A+T)/(G+C) ratios. The TMP triplet which is not populated from the exited singlet state in dilute neutral solutions can be populated by having the TMP present in high concentrations or through energy transfer from the triplet state of acetone or acetophenone. The triplet state produced in this manner has been characterized by its Δm=2 ESR line, its phosphorescence spectrum, its decay time, and the effect of deuterium substitution on the latter. In all these respects the DNA and poly dAT triplet resembles the neutral TMP triplet (T) more closely than it resembles the ionized (pH 12) TMP triplet (T−). A determination of the acidity constants of the excited states of A and T gives strong evidence against single‐proton transfer from the excited singlet or triplet state of T to A. While this cannot be considered final proof that the DNA triplet is that of neutral thymine, this is accepted as the most likely hypothesis.
Article
A model for DNA molecules is introduced and treated by the methods of statistical mechanics. This model takes into account the difference in bonding free energy between adenine—thymine and guanine—cytosine base pairs. The model used is an extension of the Zimm—Bragg model for polypeptides. The partition function for a certain class (which probably corresponds to most DNA sequences) of molecules is evaluated numerically and the linear dependence of the temperature of half‐denaturation on base composition is obtained, in agreement with the experimental work of Marmur and Doty. A further, previously unnoted, result is that the breadth of the transition is a function of the base composition. Comparison of the predicted broadening with available experimental data is excellent for DNA from simple organisms which probably possess only one kind of DNA molecule each. However, for complex organisms, which are known to have compositionally heterogeneous DNA, the relationship is not obeyed. This suggests the possibility of using transition breadth as a simple and rapid test for compositional heterogeneity of DNA's.
Article
Nucleic acids and their analogues raise more and more interest in research areas outside biology and biochemistry. The functionalization of these evolutionary optimized self-assembling macromolecules with metal ions widens their applicability even further. Previous efforts to incorporate metal ions site-specifically into nucleic acids focused on the covalent attachment of appropriate ligands to either the sugar phosphate backbone or the nucleobases. More recently, the use of metal-ion-mediated base pairs, i.e. the replacement of natural nucleobases by ligands, was suggested as an alternative means. This microreview discusses selected examples of metal-ion-mediated base pairs, including those that comprise unmodified natural nucleobases, and describes possible applications for the resulting metal-modified nucleic acids. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Article
Native deoxyribonucleic acid (DNA) binds mercuric ion reversibly at pH 9. There is one kind of a complex up to a bound mercury to nucleotide ratio (rb) of 0.5. Two protons are displaced per mercury ion bound. Synthetic or natural (crab) deoxyadenosylthymidine alternating copolymer is somewhat different in that the first complex saturates at one mercury per four nucleotides. The equilibrium concentration of Hg2+ as a function of rb has been measured. Denatured or single-strand DNA binds more strongly than native; for native DNA's, the binding strength increases with increasing thymidine content. The binding is somewhat cooperative. The buoyant density of DNA in a Cs2SO4 density gradient increases greatly on binding Hg2+; the quantitative relation dp/drb ≈ 0.4 g/ml-1 (0 ≤ rb ≤ 0.50). The selective binding reaction can be used to create large buoyant density differences between single- and double-stranded DNA, or between DNA's with different base compositions. Various separations, including the separation of the two halves of λb+ DNA, are demonstrated. The natural native DNA's readily come to equilibrium with Hg2+. Care must be taken to ensure good mixing on adding Hg2+ to synthetic dAT; otherwise, nonequilibrium, nonhomogeneous mercury complexes form. Mercury ion can be removed from DNA by adding a sufficiently strong complexing agent. The DNA is recovered in its original form, with no deterioration in its structure or biological activity. Several alternate structures for the DNA-Hg2+ complexes, all involving Hg2+ cross-links between complementary strands, are considered.
Article
Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH3Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by 1H and 13C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH3Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH3Hg(II) bind to N3 of thymidine in preference to N1 of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.
Article
Methylmercury(II) nitrate reacts with 9-methylguanine (9-MeGua) in water to form solid complexes of stoicheiometry [HgMe(9-MeGuaH–1)], [HgMe(9-MeGua)][NO3], [HgMe(9-MeGua)][NO3]·H2O, and [(HgMe)2(9-MeGuaH–1)][NO3]. Comparison of i.r. spectra of the solid complexes and 1H n.m.r. spectra of [2H6]dimethyl sulphoxide-soluble (ionic) complexes with spectra of analogous guanosine (Guo) complexes indicates that complexes of 9-MeGua and Guo with similar stoicheiometry have the same mode of binding of HgIIMe to the purine ring. The spectra indicate that [HgMe(9-MeGuaH–1)] and [(HgMe)2(9-MeGuaH–1)][NO3] have HgIIMe bonded to N(1) and both N(1) and N(7), respectively, and the complexes [HgMe(9-MeGua)][NO3] and [HgMe(9-MeGua)][NO3]·H2O have HgIIMe bonded to N(7) with retention of a proton at N(1). Crystals of [HgMe(9-MeGua)][NO3] are monoclinic, with a= 4.196(1), b= 15.060(4), c= 18.288(5)Å, β= 90.17(2)°, Z= 4, and space group P21/c. The structure has been solved by conventional Patterson and Fourier methods and refined by least-squares techniques to R 0.061 for 1 152 reflections collected by diffractometer. The complex has HgIIMe bound to N(7) with Hg–C 2.06(2), Hg–N(7) 2.09(2)Å, and C–Hg–N(7) 175(1)°. Mercury interacts weakly with nearby nitrate ions, with Hg 0 2.75(2) and 2.99(2)Å; the purine ring is planar with the HgIIMe group slightly tilted from this plane, Hg being –0.168(1) and the carbon atom –0.411(25)Å from the plane.
Article
Two Ag(I) complexes containing the model nucleobases 1-methylcytosine (1-MeC), 9-methyladenine (9-MeA), and 7,9-dimethylguanine (7,9-DimeG) have been prepared and studied: [Ag(1-MeC-N3)(9-MeA-N7)(H2O)]NO3 (2) crystallizes in the space group P2(1)/a, a = 11.167 (2) angstrom, b = 13.437 (2) angstrom, c = 11.520 (2) angstrom, beta = 109.79 (2)-degrees, V = 1626 (i) angstrom3, z = 4. Ag has a severely distorted trigonal-planar coordination geometry with two strong bonds (2.128 (2) angstrom and 2.120 (2) angstrom) to the nitrogens of the nucleobases and a weak bond (2.664 (2) angstrom) to a water molecule. The N-Ag-N vector is markedly nonlinear (angle at Ag 165.8 (1)-degrees). Both nucleobases and the water molecule are virtually coplanar. Intramolecular H bonding is between O(2) of 1-MeC and N(6) of 9-MeA (3.053 (3) angstrom) as well as between N(4) of 1-MeC and the aqua ligand (2.894 (4) angstrom). Structural details of the structure of 2 clearly demonstrate that the water molecule is an integral part of the "metal-modified" base pair. A second mixed-nucleobase complex of composition [Ag(1-MeC)(7,9-DimeG)NO3]2.[(1-MeC)(7,9-DimeGH)PF6].10H2O (3) has been prepared and characterized by elemental analysis and H-1 NMR spectroscopy. On the basis of the structure of 2 an alternative model to existing hypotheses on Ag-DNA interactions is put forward which considers the "insertion" of a metal-aqua entity into an existing base pair.
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Summary Repair of ultraviolet-light-induced DNA damage inStreptococcus lactis has been examined. The wild-type strain and its derivative Lac- possess a dark repair system (maximal increase in survival of 4-fold). Enzymatic photoreactivation exists in the two strains but a weaker photoreactivability was found in the Lac- derivative (4 and 2-fold, respectively). Concomitant reduction of UV-induced mutagenesis (Rifr marker) was also studied during these two repair phenomena. The absence of dark repair after saturation of photoreactivation suggests that photoreactivation is much more efficient with pyrimidine dimers as substrate.
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The binding of netropsin to 31 different natural and biosynthetic DNAs, DNA-RNA hybrids, and RNAs was studied in order to delineate the nucleic acid structural features necessary for binding. The measurements employed were spectral titrations, analytical density gradient centrifugation, thermal denaturation, equilibrium dialysis, and inhibition of in vitro replication and in vitro transcription. The major conclusions are: (a) in 0.1 m or higher salt concentrations, netropsin has a marked specificity for DNAs which contain A-T (or I-C) pairs. It binds tightly to two DNAs which contain only A-T pairs and to two DNAs which contain only I-C pairs. However, no measurable binding was found for two DNAs which contain only G-C pairs. (b) Netropsin's inability to bind to G-C paired regions is a consequence of the 2-amino group of guanine. (c) Netropsin is specific for duplex DNA; no binding was observed to five single-stranded DNAs, three helical RNAs, or two of the three DNA-RNA hybrids studied. (d) Netropsin binds to DNA by a nonintercalating mechanism, since it does not cause unwinding of supercoiled DNA. (e) Netropsin inhibits in vitro DNA and RNA synthesis by binding to the template or to the primer, or to both. (f) The closest distance between bound netropsin molecules is three base pairs. A molecular model for netropsin binding in the minor groove of DNA is proposed.
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Mung bean nuclease I is highly preferential for single stranded (denatured) DNA; the numerical value of this preference depends on the type of DNA and the reaction conditions; at 37° in the absence of Mg2+ it is 30,000-fold for T4 DNA, 65-fold for crab d(A-T)n, and less than 2-fold for biosynthetic d(A-T)n. No detectable hydrolysis of poly dG·poly dC occurs under these conditions even with a 100-fold excess of enzyme. Addition of mm Mg2+ inhibits the hydrolysis of biosynthetic d(A-T)n 13-fold, but accelerates that of denatured DNA 2- to 3-fold. The temperature coefficient of the reaction in the range 27–37° is 5-fold higher for biosynthetic d(A-T)n than for crab d(A-T)n. With native biosynthetic d(A-T)n conditions exist under which the rate of hydrolysis approaches that of denatured DNA from other sources (T4, Escherichia coli, thymus). Therefore, mung bean nuclease I does not recognize biosynthetic d(A-T)n as a typically double stranded structure. DNA of λ phage which is known to contain an A,T-rich region in the center of the molecule is specifically attacked by mung bean nuclease I at this region. The most important properties of mung bean nuclease I are (a) its ability in low concentration to remove denatured DNA from a mixture of both forms, and (b) its ability in high concentration to cleave specifically A,T-rich regions of double stranded DNA. The name "region-specific nuclease" is suggested for a new class of enzymes exemplified by mung bean nuclease I.
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The binding of aquated cis-(NH3)2PtII to polyriboadenylic acid, polyribocytidylic acid, and polyriboguanylic acid has been studied at pH 7, 37°C, and an ionic strength of 0.1. The total number of binding sites per nucleotide in each polynucleotide is one; the magnitude of the intrinsic binding constant increases in the order poly(C) < poly(A) < poly(G). The CPD binding profiles for poly(A) and poly(C) are similar, displaying a noncooperative behavior, while the binding profile for poly(G) is more complex and may be described by a number of alternative binding models. The data are consistent with the model in which the CPD binds very strongly to the four-stranded poly(G) aggregate up to v̄ values of 0.25, at which point the aggregate is disrupted and the subsequent binding affinity, perhaps to the same or a different site, is greatly reduced. This is the first binding data which supports the four-stranded model for poly(G) in solution. The binding affinity of CPD to homopolynucleotides is greater than to the corresponding nucleosides. The results of these studies are compared and discussed relative to similar investigations reported for other metal ions binding to nucleic acids.
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Complexes of the stoichiometry RHg(GuoH-1), [RHg(Guo)]NO3, and [(RHg)2GuoH-1]NO3 have been synthesized by reaction of the nucleoside guanosine (Guo) and RHgII in water (R = Me) or aqueous ethanol (R = Ph). Comparison of infrared spectra of the solid complexes and 1H nuclear magnetic resonance spectra of the complexes in dimethyl-d6 sulfoxide with spectra of Guo, [GuoH]NO3, and Na[GuoH-1]·H2O allows assignment of structures for these ambidentate ligand complexes. Deprotonation of N1 of Guo leads to coordination of RHgII at N1 in RHg(GuoH-1) and to both N1 and N7 in [(RHg)2GuoH-1]NO3. The complexes [RHg(Guo)]NO3 have RHgII bonded at N7 of the guanine base. Phenylmercuric hydroxide reacts with thyrnidine (dThd) in aqueous ethanol to form PhHg(dThdH-1)·H2O with PhHgII bonded to N3 after deprotonation of dThd. Structures deduced for these solid complexes are in agreement with those suggested in earlier ultraviolet and Raman studies of the interaction of nucleosides and nucleotides with MeHgII in aqueous solution. The uses and limitations of vibrational spectra in assigning structures to such metal nucleoside complexes are outlined.
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A single-crystal X-ray diffraction analysis on the complex (nitrato)(1-methylcytosine)silver(I) is presented. Crystals of the complex are triclinic with the following crystal data: a = 10.474 (3) Å, b = 11.141 (3) Å, c = 3.642 (1) Å, α = 97.33 (2)°, β = 95.82 (2)°, γ = 76.76 (2)°, V = 409.2 Å3, space group P1, Z = 2 [for [Ag(C5H7N3O)(NO3)], mol wt 295.0]. Intensity data were collected in the θ-2θ scan mode on an automated diffractometer. The structure was solved by Patterson and Fourier methods and refined by full-matrix least squares to an R value of 0.033. The packing motif in the crystal is that of a dimeric [Ag(1-methylcytosine)(NO3)]2 complex with each of the 1-methylcytosine residues doubly cross-linked by two Ag+ ions through the base binding sites N(3) and O(2). Propagation of this dimeric unit along the crystallographic c axis via a second type of Ag-O(2) bond and base-base overlap yields a two-stranded, cross-linked polymer with the base residues slightly out of register. An attempt is made to relate these observed properties to the binding of Ag(I) to regions of high G-C content in duplex DNA.
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The application of the Hg(II)-Cs2SO4 density-gradient centrifugation technique in the isolation of the λdg deoxyribonucleic acid (DNA) half-molecules is reported. At a molar ratio of Hg(II) to total DNA phosphate of 0.4, the buoyant densities of the iλ-containing half and the gal+-containing half in Cs2SO4 differ by 0.04 g/ml, and the two halves can be separated by preparative density-gradient centrifugation. The biological activities of the half-molecules are retained after addition and subsequent removal of mercury ion. If aggregates containing the two DNA halves are present, a trimodal buoyant pattern is observed with the aggregates being the middle band.
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Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated m calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity m native and in reassociated state. Two of the satellite conponents, with densities of 1.722 and 1.714 g/cm3, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm3 appear to be heterogeneous.
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Several species of a genus Cancer have a DNA that has a light buoyant density and that contains mainly deoxyadenylate and thymidylate. The presence of this polymer was demonstrated previously in testes and vas deferens. By a modified procedure for isolating DNA, the muscles, liver, and eggs of Cancer borealis are also shown to contain the deoxyadenylate-thymidylate-like polymer.
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The complexes of silver(I) with DNA have been studied spectrophotometrically and potentiometrically at an ionic strength of 0.1 M (NaClO4) and pH ∼ 5.6. Let rb be the moles of Ag+ bound per DNA base. There is one complex for 0 < rb < 0.2, with a characteristic spectrum and a high binding affinity for Ag+. There is a second complex for 0.2 < rb < 0.5 with a different spectrum and somewhat weaker binding. Still higher complexes form at higher concentrations of Ag+, but we have not studied these extensively.The Ag-DNA complexes have about the same high intrinsic viscosity as the uncomplexed DNA, indicating that the helical structure is not disrupted. There is no proton release from the DNA upon formation of the first complex. Upon formation of the second complex, there is some proton release which is greater for AT-rich DNA's.We propose that the first complex involves addition of Ag+ to the sigma electron pair of N-7 or N-3 of the purines in alternate base pairs. In the second complex, Ag+ is added to the remaining base pairs; for AT pairs, it replaces the N-1 (adenine) to N-3 (thymine) hydrogen bond; for GC pairs, it again adds to N-7 or N-3 of the guanosine.The complexing behavior of AT polymer is quite different from that of the natural DNA's.
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A chromatographic column consisting of kieselguhr impregnated with methylated albumin, for fractionating nucleic acids by their molecular size, has been reported (Mandell & Hershey, 1960; Hershey & Burgi, 1960). A simplified form of the technique using stepwise elution revealed more properties of the column, such as recognition of base composition and of hydrogen bond content of DNA. It is found that DNA with greater guanine—cytosine content is eluted at lower salt concentration. Furthermore, thermally denatured DNA is eluted in higher salt concentration than is native DNA. Using this method, a naturally existing “deoxypolyadenme-thymine” in a marine crab (Cancer borealis) was isolated from the main DNA of the animal. The method has also been applied to fractionate RNA, and it was found that it not only separates soluble and ribosomal RNA (Mandell & Hershey, 1960), but also 16s and 23s ribosomal RNA.
Article
I have constructed a step-function binding theory for the reaction of Hg(II) and double-stranded polynucleotides. The theory specifies 2:1 base-Hg(II) complexes which bridge the double helix as the sites of the reaction when the ratio, r ≡ [Hg(II) added]/[base] ≦ 0.5. Several kinds of evidence indicate tthat Hg(II)-(thymidine)2 complexes are the most readily formed. Necessarily, the theory stipulaes perturbation of the Watson-Crick helix in reacting with Hg(II). The minimal perturbation is taken to be an axial chain shift by one base spacing. Combined with stoichiometric data on the complexing of Hg(II) and nucleosides, the chain-shift mechanism enables a quantitative evaluation of the average number of hydrogen ions released by a double-stranded polynucleotide in the interval r = 0 to r = 0.5. The theory largely explains the seeming anomaly in the mercurimetric pH-stat titration curve of highly ordered A-T polymer compared to natural DNAs. Since the chemical mechanism is the same for both A-T polymer and the natural DNAs, the reaction should prove useful in distinguishing between order and disorder in the base sequence of polynucleotides. I have also briefly discussed the application of the reaction to some aspects of the physical chemistry of polynucleotides and to the determination of their base composition and fractionation.
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