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At least 19 recordsLinked to original sources
PubMedDate not supplied

[Molecular melting of DNA and the effect of the fine structure of fusion curves].

The progress in understanding the phenomenon of molecular melting (helix -- coil transition) of DNA is considered. It is shown that the theory of DNA melting has reached such a stage of development when it is capable to compute melting profiles and denaturation maps for DNA with any given nucleotide sequence, on the base of a rather simple but adequate model of DNA. The effect of fine structure of the DNA melting profiles, its origin and possible applications are considered in greater detail. It is demonstrated that a direct comparison between experimental and theoretical melting profiles for the open replicative form of the upsilon X-174 phage DNA for which the complete nucleotide sequence has been published recently, confirms the theory and opens new possibilities for further investigation of equilibrium and kinetic properties of DNA molecules.

Bacteriophages↗
PubMedDate not supplied

[Effect of single-stranded and double-stranded breaks on the melting temperature of phage T2 DNA].

The effect of single- and double-stranded breaks in DNA phage T2, on the melting temperature of this DNA in the 0,05 M SSC solution, was investigated. The number of cleavages per 1000 nucleotide pairs varied in the range of 0 to 10. It is shown that single- and double-stranded breaks affect the melting temperature with approximately (within 20%) the same efficiency. The relationship between the melting temperature shift (delta Tm) and the number of cleavages is non-linear. The magnitude of the effect is characterized by delta Tm of 2 +/- 0.4 degrees C for the average inter-cleavage distance of 200 base pairs. It is shown that the observed melting curves are non-equilibrium ones, which is probably due to the fact that the effect of cleavages on the melting temperature is largely results from the complete and practically irreversible separation of strands.

Coliphages↗
PubMed1978

A calorimetric investigation of melting of tRNAAsp from brewer's yeast.

The thermodynamics of tRNAAsp unfolding was studied using a precision scanning microcalorimeter. The overall heat of melting was found to be about 55 J/g irrespective of the ionic strength and magnesium activity. The analysis of complex melting curves obtained in the absence of Mg2+ reveals four successive two-state transitions. The first was identified as the cooperative melting of the tertiary structure and the D region and the others as the melting of individual helical arms.

Aspartic Acid↗
PubMed1977

Calorimetric studies on melting of tRNA Phe (yeast).

The heat effects involved in thermal unfolding of tRNAPhe from yeast have been determined in various buffer systems by direct differential scanning calorimetry. Perfect reversibility of the melting process has been demonstrated for measurements in the absence of Mg2+ ions. The overall molar transition enthalpy, delta Ht = 298 +/- 15 kcal mol-1 (1247 +/- 63 kJ mol-1), has been shown to be independent of the NaCl concentration and the nature of the buffers used in this study. Delta Ht is identical in the presence and in the absence of Mg2+ ions within the margin of experimental error. This experimental result implies a vanishing or very small heat capacity change to be associated with melting. Decomposition of the calorimetrically determined complex transition curves, on the assumption that the experimental melting profile represents the sum of independent two-state transitions, results in five transitions which have been assigned to melting of different structural domains of the tRNA.

Calorimetry↗
PubMed1979

[Melting and reassociation of double-stranded RNA of the encephalomyocarditis virus].

The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.

Encephalomyocarditis virus↗
PubMed1978

Denaturation of mouse satellite DNA upon melting of chromatin in solution.

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.

Animals↗
PubMed1978

Spectral analysis of high resolution direct-derivative melting curves of DNA for instantaneous and total base composition.

Derivative melting profiles of DNA have been obtained directly by recording the difference in absorbance between two identical solutions maintained at a small constant temperature differential. This deltaA is monitored continuously with increasing temperature in a ratio recording spectrophotometer. Resolution of complex hyperfine structure in the profiles of small homogeneous viral DNAs appears to be significantly better than has been produced by various numerical methods of differentiation. In addition, a spectral method has been modified that permits easy analysis for DNA base composition from the ratio of derivative melting curves obtained at 282 and 260 nm. Eight bacterial and three vertebrate DNAs have been analyzed for total base composition from the product of the instantaneous base composition at small temperature intervals (0.05 degrees C) throughout the entire melting region and the integrated area of the 282 nm profile. The results are in excellent agreement with values determined by traditional methods.

Bacillus subtilis↗
PubMed1979

DNA sequencing and melting curve.

The dependence of DNA absorbance (for light at about 260 nm) on temperature is related to a specific DNA sequence structure in the vicinity of DNA thermal denaturation (the so-called DNA melting or coiling). A straightforward analysis of the experimental DNA melting curve allows us to determine the lengths, the A+T content, and the location in DNA of certain domains. In the case of a specific DNA fragmentation, the order of fragments in DNA can be learned from this analysis, nondestructively and quickly, without fractionating the fragments and other methods of fragmentation. If the DNA nucleotide sequence is known except for some sites and uncertain portions, the analysis determines these sites and the accuracy of the sequence at the portions. This information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage phiX174, whose melting curve is known. The results are compared to and found to be in an excellent agreement with the known phiX174 nucleotide sequence.

Base Sequence↗
PubMed1979

[On the polymorphism of barbiturates in powders and tablets. Part 1: Crystal optical studies on, and melting behaviour of barbital (author's transl)].

Crystal optical studies showed that barbital brands that conform to the standards of the DAB 7--DDR and the Ph. Hung. VI, respectively, present two different crystalline forms. After recrystallization form water or acetone-water mixture (1:1), the optical crystal picture of the brand conforming to the DAB 7--DDR prevails. The melting points of the commercial products do not differ from each other; both substances melt at 187.5 degrees C, whereas the melting points of the recrystallized products are 1.5 degrees and 2 degrees C, respectively, lower.

Barbital↗
PubMed1977

[Structural characteristics (reassociation and melting kinetics) of kinetoplast DNA from Crithidia oncopelti].

A highly purified associate of kinetoplast DNA is isolated from C. oncopelti, and its physico-chemical properties are studied. Both native associate and its ultrasonic fragments are found to have a complex character of melting. 5-6 melting zones (3 of them being the main) are found on the melting curve. Analysis of reassociation kinetics of sonicated associate of kinetoplast DNA has revealed the presence of at least two components: fast reassociating component (65-70% of complex DNA), which reassociation kinetics is equivalent to the unique sequence with molecular weight of 2.3. - 10(6) daltons, and slow reassotiating component (15% of complex DNA), having reassociation kinetics equivalent to unique sequence of 26 - 10(6) daltons. The data obtained suggest that complex associate of kinetoplast DNA is heterogenous for its nucleotide sequence and base composition.

Animals↗
PubMed1975

In vivo and in vitro aging of collagen examined using an isometric melting technique.

1. In vivo and in vitro aging of tendon from rat tail, kangaroo tail and human wrist tendon was examined by the technique of isometric melting, in physiological saline. 2. For all these collagens, two mechanisms of structure stabilisation can be distinguished in the melting curves. One of these involves co-valent cross-linking as judged by its increasing stability to heat and acid pH, while the second appears to involve only secondary interactions. 3. The time rate of the first process is slow in vivo; rat tendon up to 2 years does not show it, but it is present in 6-year-old human tendon. However, its in vitro rate is markedly dependent upon the free oxygen content of the physiological saline. At an oxygen concentration of 300 nmol/ml, the in vitro aging rate is about 30 times the in vivo rate for rat tail tendon, and about 20 times for both kangaroo tail tendon and human wrist tendon. At a concentration of 60 nmol/ml (which is about the same as normal arteriovenous blood difference) in vitro aging proceeds close to the in vivo rate.

Age Factors↗
PubMed1979

Melting-profile analysis of thermal stability of thermolysin. A formulation of temperature-scanning kinetics.

The melting-profile method consists of a continuous observation of a structural parameter while the temperature of the sample is raised at a constant rate [Fugita, S. C., & Imahori, K. (1974) IN Peptides, Polypeptides and Proteins (Blout, E. R., Bovey, F. A., Goodman, M., & Lotan, N., Eds.) p 217, Wiley, New York, N.Y.]. An analytical solution to the melting profile was formulated for the two-state irreversible process and called temperature-scanning kinetics. The theory was tested with thermolysin with consistent results, and the thermodynamic parameters of thermal denaturation were calculated: deltaH identical to = 80.3 kcal/mol, deltaS identical to = 153 eu. These values agreed with the corresponding values obtained from the classical constant-temperature relaxation kinetics. The possibilities of temperature-scanning kinetics are discussed.

Calcium↗
PubMed1979

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.

Kinetics↗
PubMed1975

The DNA melting transition in aqueous magnesium salt solutions.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).

Animals↗
PubMedDate not supplied

[Nature of the easily melted portions of DNA with a quasi-random base sequence].

The influence of denaturation conditions upon the character of partial denaturation of DNA with random base distribution were thoroughly studied. Maps of partial DNA denaturation were obtained at T less than TAT for phage phiB DNA at pH 10.7 and 5.5; Tg9 DNA at pH 8.8; at T less than TAT for phiB DNA at pH 10.9 and Tg9 DNA at pH 8.8. The map quality was better when obtained at higher pH values; the peaks became sharper and higher against the background. We failed to obtain maps of partial denaturation at pH 5.5, T less than TAT. The improvement of the map quality and existence of the partial denaturation maps at T less than TAT at pH 10.9 were explained by the increase of primary melting probability of AT-rich DNA regions. At high pH the denaturation map quality was temperature independent. This was explained by a very weak temperature dependence of primary melting probability for all maps of equal quality. The map quality became worse, when the quantity of loops was increased.

Base Sequence↗
PubMed1977

Synthesis and thermal melting behavior of oligomer-polymer complexes containing defined lengths of mismatched dA-dG and dG-dG nucleotides.

Model DNA polymers containing heteroduplex regions of defined sequence and size were synthesized using polynucleotide phosphorylase and calf thymus terminal transferase. Heteroduplexes were of the form (dG)n-d(C12AmC-x), where m - 1-6, and (dG)n-d(C10GmC-x), where m = 1 and 3-5. Thermal melting studies of the model DNAs indicated that the heteroduplex regions did not disrupt the cooperative interaction between the flanking regions of dG-dC base pairs. thus, it is possible that the heteroduplex nucleotides are accommodated in a stacked helical structure.

DNA Nucleotidyltransferases↗
PubMed1979

Fine structure melting of viroids as studied by kinetic methods.

The conformational transitions of five viroid species were studied by melting analysis and by fast and slow temperature jump techniques. Experiments with the fast temperature jump technique had to be carried out in 10 mM Na-cacodylate, 0.1 M NaCl, 4 M urea, 1 mM EDTA, pH 6.8. In addition to the highly cooperative main transition (Tm between 46.5 and 49 degrees C for different viroid species [1]) all viroids show at higher temperatures an intermediate transition (Tm approximately equal to 57 degrees C) and a high temperature transition (Tm approximately equal to 68 degrees C). The maximum amplitudes of these transitions amount only to about 1% of that of the main transition. The main transition represents a net dissociation of 78 to 94 base pairs depending on the viroid species. The intermediate transition corresponds to the dissociation of two hairpins with 5-10 base pairs each, and 10-20 nucleotides in the loops. The high temperature transition corresponds to a hairpin of 9 G:C pairs and 1 A:U pair and more than 40 bases in the loop. It is shown that these stable hairpins are not part of the native structure but are newly formed during the main transition. Their formation is responsible for the extraordinary cooperativity observed in the main transition. Hairpins can be correlated to defined sequences of PSTV. Based on these studies, on the sequence of PSTV [2], and on a theoretical treatment [3] a detailed description of the whole mechanism of PSTV denaturation is given.

Base Sequence↗