Intermediates in the strand-separation transition of T7 DNA.

Sedimentation velocity runs as a function of temperature in the region of the alkaline helix-coil transition have enabled us to demonstrate the existence of stable two-stranded intermediates in the strand-separation process for T7 DNA. The strand-separation transition under these conditions has an intrinsic breadth of ～1°C, and within this temperature range (T_m + 2°C < T < T_m + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single-stranded DNA. Parallel characterizations of the strand-separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA

The optical density–temperature profile of double-stranded poly(A + U), triple stranded poly(A + 2U), and double-stranded RNA from rice dwarf virus in solutions with and without poly-L -lysine has been examined. When poly-L -lysine is added, more than one melting temperature T_m is observed for poly(A + U) and poly(A + 2U). One of them is considered to correspond to the melting of the polynucleotide molecule free from poly-L -lysine, and another to the melting of a polynucleotide–poly-L -lysine complex. For rice dwarf virus RNA, the T_m assignable to the complex is not found to be lower than 99°C. In every case, however, the hyperchromicity observed at the T_m of the free poly-nucleotide molecule is lowered linearly as the amount of poly-L -lysine added to the solution increases. This fact is taken as indicating that there is a stoichiometric complex formed. The stoichiometric ratio lysine/nucleotide in each complex is determined by examining the relation between the amount of poly-L -lysine added to the solution and the percentage of hyperchromicity remaining at T_m of the free polynucleotide molecule. The ratio is found to be 2/3 for all of the three complexes. A discussion is given on the molecular conformations of four types of polynucleotide–polylysine complex hitherto found: (A) double-stranded DNA plus poly-L -lysine in which the lyslne/nucleotide ratio is 1, (B) three-stranded RNA [poly(A + 2U)] plus poly-L -lysine in which the ratio is 2/3, (C) double-stranded RNA [poly (A + U) or rice dwarf virus RNA] plus poly-L -lysine in which the ratio is 2/3, and (D) double-stranded RNA [poly(I + C)] plus poly-L -lysine in which the ratio is 1/2.     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Variation in nuclear DNA base composition (mol % G + C) in three orders of marine green algae

Estimates of nuclear DNA base pair composition by determination of thermal denaturation temperatures (T_m) indicated guanine + cytosine (G + C) levels of 35–56% for 17 species of marine green algae. T_m values were found to be reproducible with coefficients of variation among samples and replicates of generally less than 1 percent. G + C % values in four species of Enteromorpha varied within a narrow range of 53–56%, whereas values for three species of Ulva showed substantially greater variation, ranging from 35–55%. Ulva fasciata collections from two geographically separate North Carolina sites had mean G + C composition of 44.8 and 35.6 respectively, suggesting that these populations may be genetically distinct. Enteromorpha linza, which has been treated as a species of Ulva, had a G + C composition of 53.2, typical of the Enteromorpha species investigated. Nuclear DNA base pair composition data for species of Cladophorales and Caulerpales are given as well.Center for Marine Science Research, UNC-W contribution No. 009.     

Determination of DNA base compositions from melting profiles in dilute buffers

Equations were determined for the dependency of the melting temperature (T_m) of DNA upon the logarithm of the sodium ion concentration, for four DNA samples of widely different base compositions (θ_GC). The slopes of these T_m versus log M equations wore found to decrease with increasing θ_{G C}of the samples. An empirical equation relating T_m, log M (Na⁺) and θ_{G C} was derived, which also accounts for differences in T_m versus log M slopes. Data from the literature for some synthetic polynucleotides and for the crab(Cancer pagarus) satellite poly AT are discussed in relation to the above finding. The changes in T_m versus log M slopes with θ_{G C} are interpreted in terms of changes in the thermodynamic parameters ΔS and ΔH with base composition.     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis: renaturation and sedimentation studies

The renaturation kinetics of mitochondrial DNA from the yeast Saccharomyces carlsbergensis have been studied at different temperatures and molecular weights. At renaturation temperatures 25 deg. C below the mean denaturation temperature (T_m) in 1 M-sodium chloride the renaturation rate constant is found to decrease with increasing molecular weight of the reacting strands. This unusual molecular weight dependency gradually disappears with an increase in the renaturation temperature. At a temperature 10 deg. C below the melting point, the rate constant shows the normally expected increase with the square root of the molecular weight. From the renaturation data at this temperature, the molecular weight of the mitochondrial genome is estimated to be about 5·0 × 10⁷. The same size of genome was found from renaturation at low molecular weight and 25 deg. C below the T_m.The sedimentation properties of denatured mitochondrial DNA at pH values 7·0 to 12·5 were used to study the conformation of this DNA in 1 M-sodium chloride. The results obtained support the conclusion from the renaturation studies: that the pieces of denatured mitochondrial DNA with a molecular weight above 2 × 10⁵ to 3 × 10⁵, in 1 M-sodium chloride at 25 deg. C below the mean denaturation temperature are not fully extended random coils. Presumably, interaction between adenine and thymine-rich sequences, which are clustered at certain distances within the molecules, is the molecular basis for these observations.     

Synthesis of Polyoxamic Acid (2-Amino-2-deoxy-l-xylonic Acid

The rate of precipitation of the retrograded amylose product from a dil. amylose solution was determined by the centrifugal method. The results showed that the relation of the quantity of precipitate vs. time did not fit the typical second order reaction for the coalescence of colloidal particles but fitted the crystallization formula, in appearance.

The rate of precipitation was in proportion to (c-c_a)^1.5, where c is the amylose concentration and c_a the concentration of the dil. solution phase in the phase-separated solution. When the temperature dependence of the rate was treated according to the crystallization of polymers, it was found that the rate was in proportion to T_m²/T(ΔT)², where T_m is the melting point of the polymer in solution and ΔT is (T_m?T). The T_m thus obtained was 120°C for an amylose solution. These results suggested a certain correlation between the amylose retrogradation and the crystallization.     

Improvements in allelic discrimination of microsatellite markers using denaturing polyacrylamide gel electrophoresis

Poor resolution, retarded progress of DNA through gels, and variable sizing of DNA fragments between and within gels hinder accurate genotyping of some simple sequence length polymorphism (SSLP) markers with the Perkin Elmer Applied Biosystems 377 Sequenator. These problems are similar to renaturation related problems observed in DNA sequencing gels. PCR products especially susceptible to these problems are shown to have higher melting temperatures (T_m) than others. Gels containing increased concentrations of denaturants allow greater accuracy in allelic discrimination. This is especially beneficial where quantification is necessary. Received: 7 February 2000 / Accepted: 16 March 2000     

Hetero-α-helical,two-chain coiled-coils. Clam–worm hybrid paramyosins

The specificity of the interaction between the α-helices in two-chain coiled-coils is investigated by studying the formation of hybrid molecules in which one α-helix is a clam paramyosin chain and the other a worm paramyosin chain. Hybrids are formed by mixing, denaturation, and subsequent renaturation. Comparison is made with a blank solution in which renaturation precedes mixing, thus precluding hybridization. Hybrids are detected by a ruse based on the presence of free sulfhydryl functions on calm chains. This allows molecules comprising two clam chains to be covalently crosslinked by oxidation with 5,5′-dithiobis(2-nitrobenzoate). Worm paramyosin chains have no sulfhydryl, so molecules comprising two worm chains or hybrid molecules comprising one chain of each type cannot crosslink. When run on sodium dodecyl sulfate polyacrylamide gel electrophoresis, therefore, the protein separates into two well-resolved regions, one containing one-chain species and the other two-chain species. When the gels are scanned and quantitated, the hybrids show up as an increase in the fraction of material in the one-chain band compared with the fraction in the blank solution. When renaturation is direct, we find that the fraction of renaturated molecules that are hybrids varies from ～10% at 5°C to ～5% at 25°C. These are judged to be nonequilibrium (quenched) values. When renaturation is by slow annealing, the equilibrium fraction hybrids are ～4% and show a modest, but measurable, increase with increasing temperature. These data allow calculation of the equilibrium constant K_h and standard free energy for the hybridization reaction: (1/2)CC + (½)WW = CW, in which C(W) stands for an α-helical clam (worm) polypeptide chain. The temperature dependence gives the standard enthalpy and entropy of the reaction. We find ΔH ? 1800 cal mol^?1 and ΔH ? 1.4 cal mol^?1 K^?1, using molarity concentration units and the infinitely dilute solution in NaCl/phosphate buffer as reference state. The possible molecular significance of these values is discussed, and it is concluded that the observed standard entropy arises essentially entirely from the rotational dissymmetry of the hybrids.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Thermal Stability of Immobilized Glucoamylase Entrapped in Polyacrylamide Gels and Bound to SP-Sephadex C–50

Glucoamylase[α-1,4: 1,6-glucan-4: 6-glucohydroease, EC 3.2.1.3] from Rhizopus niveus was entrapped in polyacrylamide gels and adsorbed onto SP-Sephadex C–50 to elucidate the thermostability mechanism of immobilized enzymes. The thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels was enhanced slightly compared with glucoamylase in free solution, and was independent of the acrylamide monomer concentration and N, N′-methylene-bis (acrylamide) content. To explain this phenomenon, the cellular structure of polyacrylamide gel was taken into consideration in addition to interactions between glucoamylase and gel, and a decrease in dielectric constant in the gel [S. Moriyama et al., Agric. Biol. Chem., 41, 1985 (1977)¹⁾]. On the other hand, immobilized glucoamylase bound to SP-Sephadex by ionic interaction showed lower stability than free glucoamylase, and much greater stability than glucoamylase in the presence of dextran sulfate, a constituent of SP-Sephadex. Thermal stabilities for the free and immobilized enzymes were also compared at the pH not in the bulk solution, but in the SP-Sephadex.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity as previously speculated.     

Correlation of Tm and sequence of DNA duplexes with delta H computed by an improved empirical potential method

T_m values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH _calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between T_m and ΔH _calc (correlation coefficient, 0.99), with T_m values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH _calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate T_m and (relative) ΔH _calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔH_calc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that T_m of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

DNA of Akodon (Rodentia,Cricetidae). I. Biophysical characterization

Buoyant density in CsCl, melting temperature, and G + C base content of the DNA from four species of Akodon (Rodentia, Cricetidae) were determined. The buoyant density values of 1.699–1.701 g/cm³ were in accordance with the data reported for other cricetids. No satellite bands were seen in neutral CsCl. The T _m values determined in 1 × SSC ranged from 86.2 to 87.0 C, which corresponds to G + C contents of 41.2–43.2%. There was good agreement in DNA base composition of the four species, although values were slightly higher in A. obscurus, suggesting a certain degree of interspecies variability.This study was supported by grants from Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, and Organización de Estados Americanos.     

The satellite bands of the DNA of Drosophila virilis

Purified DNA has been prepared from Drosophila virilis using a modification of the method derived for bacteria (Marmur, 1961). Some physical properties have been examined, a new hidden satellite discovered, and a difference found in the satellite banding pattern of different tissues. — In addition to the three satellite bands lighter than the main band previously reported (Gall et al., 1970), a new satellite heavier than the main band has been detected after thermal denaturation of the DNA (which substantially shifts the buoyant density of the main band but not that of the satellites indicating that all are fast-annealing). The satellite pattern of DNA extracted from heads alone differed from that of the entire animals: the amount of satellite I was decreased and II increased; III was unaffected; IV was increased relative to the amount in the main band. The total content of satellite material in the heads (assumed to be entirely diploid) was 42%, the highest amount reported for any organism. — Thermal transitions were determined for the DNA from adults and larvae. After preparative CsCl density gradient fractionation of adult DNA, two sets of bimodal thermal curves were obtained (in SSC) with agreement between the initial position in the preparative gradient, the thermal transitions, and the G+C content from density except for satellite III for which the T_m gave a more accurate G+C amount. DNA from satellites I and II together generated a T_m of 81.2° which was similar to a calculated T_m of 81.9° making the naive assumption that the thermal components of the two satellites would interact in a simple additive fashion. A T_m of 71.9° was ascribed to satellite III which indicates that it is not the equivalent of the poly (A-T) band found at the same density in D. melanogaster (Fansler et al., 1970). The calculated overall base composition from the density equivalents (using the value for satellite III from thermal data) gave an expected G+C content of 36.6%. The measured value was 36.0%. The possible significance of the differential satellite pattern has been discussed.