The effect of nucleotide substitution on DNA denaturation profiles.

The melting profiles were obtained for DNA restriction fragments of approx. 1150 bp with deletion of one, five or six base pairs making them different from each other. In all cases the deletions caused a shift of one melting peak without affecting the positions of the other three peaks. The effect amounted to 0.28 +/- 0.03C upon the deletion of one GC pair. The melting of DNA fragments was also studied by electrophoresis in denaturing gradient gels. The deletion of one GC pair was shown to cause an appreciable shift of the electrophoretic denaturation profile.     

High resolution thermal denaturation of DNA: thermalites of bacteriophage DNA.

High resolution thermal denaturation profiles are presented for the DNAs of bacteriophages lambda and T7. It is concluded that the temperature increment in data gathering and the method of calculating results meet the requirements for quantitative recording of the large amount of information found in the thermal transitions of both DNAs. The high resolution derivative denaturation profiles of these bacteriophage DNAs demonstrate that individual subtransitions (thermalites) of natural DNA are Gaussian in form and have narrow transition widths. Curve resolution performed on these profiles indicates that the mean thermalite width (2 sigma) is 0.33 degrees C and that this breadth is relatively invariant. Transition widths are not influenced by the position of thermalites in the profile or by cation concentration in the range from 5 to 30 mM Na+. However, the relative position of thermalites within a denaturation profile is a function of the solution ionic strength. The distribution of lengths of the DNA sequences which these thermalites represent is broad, with a number average length of 900 base pairs. Although we find an approximate similarity between the number of thermalites in the denaturation profile of T7 DNA and the number of looping regions in the electron microscopic partial denaturation map of Gomez and Lang ((1972), J. Mol. Biol. 70, 239-251) we conclude that free solution thermal denaturation experiments can be compared only superficially to the mapping results.     

The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.     

Effects of thermal denaturation on protein glycation

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.     

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.     

Thermal fluctuation spectroscopy of DNA thermal denaturation

We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak.     

Theoretical melting profiles and denaturation maps of DNA with known sequence: fdDNA.

Differential melting profiles and denaturation maps are calculated for fdDNA whose sequence of nucleotides has been determined recently. The melting profiles for the total DNA and a number of its restriction fragments are compared with experimental data taken from literature. The comparison enables one to correlate a number of peaks on experimental melting profiles with the melting out of concrete regions of the nucleotide sequence. For three fragments very strong end effects are demonstrated on both theoretical and experimental profiles. These anomalous end effects are shown to be connected with a region highly enriched with AT-pairs. A possible influence of the heterogeneity of stacking interaction on the results obtained is discussed in detail.     

Similarity of genetic distance determined from DNA thermal denaturation profiles to standard estimates of bacteriophage relatedness

High resolution thermal denaturation profiles of a series of potentially related bacteriophages of Bacillus subtilis contain a multitude of distinctive features that permit them to be divided into at least four groups. In addition, the profiles can be used to derive the quantitative parameter, genetic distance, defined by Soumpasis (Soumpasis, D. (1980) J. Theor. Biol. 86, 137-147). Both the quantitative and the qualitative intercomparisons of profiles are in general agreement with the results of standard techniques for estimating genetic relatedness.     

Genetic distance in fungus genus Fusarium measured by comparative computer analysis of DNA thermal denaturation profiles

Summary High resolution thermal denaturation profiles of different members of fungus genus Fusarium were compared with respect to the shape of their DNA melting curves. Quantitative comparison of the shape (areas under differential curves) of all thermal denaturation profiles was made. Thermal denaturation profiles can be used to derive the quantitative parameter, genetic distance, defined by Soumpasis (12). Based on such data of genetic distance a dendrogram and a genetic distance tree was constructed.     

Effects of base damages on DNA replication.

Understanding the response of DNA polymerase to the encountered damage in a template is a key to assessing lethal and mutagenic events of cells exposed to genotoxic agents. In the present study M13 (or f1) DNA templates containing 4 types of thymine damages were prepared, and DNA synthesis was carried out in vitro with the templates. The extent of inhibition of DNA synthesis by the damages was evaluated by measuring [3H]dTMP incorporation. Furthermore, newly synthesized DNA was analyzed on a sequencing gel to determine termination sites of DNA synthesis. The results showed that DNA synthesis was differentially inhibited by the damages, and the termination sites of DNA synthesis were dependent on the structures of the damages and the 3'-5' exonuclease activity of DNA polymerase used.     

High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions.

The fidelity achieved in first derivative profiles of DNA thermal denaturation is shown to depend on a number of factors including the thermal increment of data gathering, the precision of absorbance readings, and the manner in which data are smoothed prior to calculating the derivative of hyperchromicity. The closeness with which thermal denaturation data can be fitted by a cubic polynomial is carefully considered, and a derivation is presented for the estimated error in calculated values of the derivative of hyperchromicity with respect to temperature. After reviewing both theoretical and experimental evidence for the expected minimum width of a thermal transition in DNA, we conclude that thermal increments of 0.05°C or less are required for an adequate representation of transitions in naturally occurring DNA's. Data gathered under conditions meeting the requirements suggested here for quantitative recording of thermal denaturation profiles (Vizard and Ansevin, submitted for publication) show that virtually all of the high-resolution thermal denaturation profile of a simple, naturally occuring DNA may consist of small subtransitions, which we call thermalites. The finding of substransitions is consistent with current theories of DNA melting. A particularly well-resolved thermalite of λ bacteriophage DNA has a breadth of only 0.30°C (2σ width), and thus is narrower than previously reported thermal transitions for DNA. For this thermalite, the combination of width, shape, and position in the profile suggests that the substransitions observed in accurately recorded DNA thermal denaturation profiles are not described satisfactorily by existing theories. Knowledge of the requirements for the quantitative recording of thermal denaturation profiles should greatly favor the usefulness of denaturation experiments for physical genomic analysis.     

Deep-freezing of bacterial DNA for thermal denaturation and hybridization experiments

What effect freezing purified DNA at −70 C and then keeping it at −21 C for several months might have on the thermal denaturation and hybridization was investigated. One part of each DNA sample was stored at 4 C, and the other frozen at −70 C and stored at −21 C, and the results of thermal denaturation and hybridization experiments were compared. They show that freezing at −70 C and then storing at −21 C for half a year or probably even one year do not significantly affect thermal denaturation and hybridization.     

The thermal denaturation of DNA: average length and composition of denatured areas

A spectral study of melting curves of DNA ranging from 73 to 32% AT indicates that the base ratio of sequences melting within DNA are a linear function of temperature. A study of partially denatured DNA by electron microscopy, reversible renaturation and fractionation on hydroxylapatite suggests that the melting curve of DNA represents the melting of sequences which average 3-4 million daltons in length. These sequences appear to be a combination of two areas, one which is high in AT and denatures in the first three-quarters of the melting curve, and one which is high in GC and denatures in the final quarter. The length of these sequences appears to vary between 1.5-6 million daltons.