Thermal Stability Of DNA Research Articles

Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. CHTI 55 and its effect on the thermal stability of DNA

In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) ( M r 10760) and MC2 ( M r 17000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.

Calibration of the change in thermal stability of DNA duplexes and degree of base pair mismatch.

One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (delta Tm) to base pair mismatch have led to conflicting results. We have studied the delta Tm between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of delta Tm and percent base pair mismatch is remarkably linear over this range with a correlation coefficient greater than 0.98. A delta Tm of 1 degree C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The mmelting temperature of duplex DNA is lowered by 0.6°C per per cent of formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30% it does not affect the rate of DNA reassociation.

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

AbstractThe 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].

Mechanisms of polyanion stimulation of nuclear DNA synthesis: Examination of thermal destabilization

The polyanions heparin and poly- l-glutamic acid stimulate endogenous DNA synthesis in isolated nuclei and chromatin and destabilize the 78000 g fraction of rat liver chromatin to thermal denaturation; however, there is a poor correlation between the polyanion concentrations that result in large-scale template destabilization and those that stimulate endogenous DNA synthesis. We have isolated a chromatin fraction that is enriched in endogenous DNA synthesis, the degree of enrichment being significantly greater with heparin-treated chromatin; however, in both treated and untreated chromatin this fraction is indistinguishable from the remaining chromatin by thermal denaturation in dilute EDTA. This fact, as well as the different polyanion concentrations required to alter endogenous DNA synthesis and thermal stability, leads us to conclude that large-scale template destabilization does not correlate with increased endogenous DNA synthesis.

Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea.

This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lower the Tm by 2.25 degrees C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.60 degrees C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.

In Vitro Iodination of Plant Ribonucleic Acids

The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 x 10(4) to 10 x 10(6) cpm/mug. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and (125)I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between (125)I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [(32)P]chloroplast-rRNA. Thermal stability of DNA-(125)I-rRNA hybrids was similar to the thermal stability of DNA-[(32)P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties.

Age-dependent increase of thermal stability of in situ chromatin of rat liver and its reversal after hepatectomy.

An age-dependent increase of thermal stability of DNA in situ has been demonstrated in rat liver by means of microfluorimetry, which was reversed to a great extent in old regenerated liver.

DNA homologies among Drosophila species and a related genus.

Deoxyribonucleic acids of members of the family Drosophilidae; e.g., D. melanogaster, D. robusta, D. pellewae, D, immigrans, D. mcclintockae, D. calloptera, C. procnemis and from a tissue culture cells of D. melanogaster have been compared with respect to base composition, heterogenecity, and nucleotide sequence homology. Considerable heterogeneity exists in DNA's from 3rd instar larvae and tissue culture cells. The DNA base composition of adult species ranges from 33-42 moles percent GC; in addition a polydAT component is apparent in larval DNA's. There are about 21 and 29 percent intragenomic homology in DNA's of D. melanogaster and D. immigrans, respectively. Relatively large differences were revealed in the nucleotide sequences of several species by DNA hybridization and thermal stability studies.

Thermal Stability of DNA: DNA Hybrids within the Genus Agrobacterium

SUMMARY: Molecular hybrids were prepared between unlabelled DNA from representative strains of eleven genetic races of Agrobacterium and [14C]DNA from typical strains of each of the three main races. The thermal stability of each hybrid was determined. The nature of the hybrids formed varied with the incubation temperature and the kind of DNA used. Hybridization in 2 × SSC-30% dimethylsulphoxide below 59°C yielded two kinds of hybrids: a labile one of unknown nature, denaturing below 59°C, and a more or less stable hybrid denaturing above that temperature. The latter was the only one formed in hybridizations at or above 59°C. There were three kinds of stable hybrids. Within each of the main Agrobacterium races thermal stability of the molecular hybrid was about the same (within 2°C) as for the homo-duplex. Between two races of 50% DNA relatedness, the duplexes were about 6°C less stable. Between races of 10 to 15% DNA relatedness, the duplexes were weak, and the stability was at least 13°C lower. The stability of the hybrids decreased concomitantly with the degree of DNA relatedness. The decreased hybrid denaturation curve is not due to AT-rich sequences. The less two races of agrobacteria appeared to be evolutionarily related, the more mutations occurred within the common part.

Relatedness between major taxonomic groups of fungi based on the measurement of DNA nucleotide sequence homology.

A procedure for the isolation of highly purified labeled and unlabeled DNAs from fungi representing three major groups has been described. The yield of DNA per g weight of freeze-dried mycelia is always higher inNeurospora crassa thanCoprinus lagopus andMucor azygospora. Thermal melting profiles show that theN. crassa andC. lagopus DNAs have one low G+C (32 mole percent) and another high G+C (52 mole percent) component, whereas theM. azygospora DNA has only one low G+C (38 mole percent) component. Based on DNA: DNA reassociation kinetics studies in this and other laboratories the genome size ofM. bacilliformis, N. crassa andC. lagopus was found to be 2×1010, 2.2×1010 and 2.5×1010 daltons respectively.N. crassa32P DNA when hybridized withCoprinus andMucor DNAs gave an average of 14 and 11% homologies respectively but when hybridized with DNAs of other species of the same genus gave a homology of 83% on an average. When32PCoprinus DNA was hybridized withN. crassa andM. azygospora DNAs and average of 10 and 8% hybridization, respectively, was obtained. The DNA: DNA homology and thermal stability data indicate that the twoNeurospora species,N. intermedia andN. sitophila, are equally distant fromN. crassa and probably originated from the same ancestor in the evolutionary scale. On the basis of genome size comparison between the three fungi, theCoprinus (basidiomycetes) seems to be highly evolved whereasMucor (phycomycetes) is a relatively primitive one,Neurospora (ascomycetes) being somewhere in between these two groups in the evolutionary scale.

Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nuclei

Denaturation of double-stranded DNA into a single-stranded state can be studied by heating fixed cells attached to quartz slides and then determining the increase in nuclear UV-absorption at 265 nm by microspectrophotometry at room temperature. In order to prevent renaturation as the slides are cooled, formaldehyde is added to the solution in which heat denaturation is performed. The influence of formaldehyde concentration, duration of heating and ionic strength on the stability of DNA to heat denaturation has been examined. The standard method involves heating of ethanol/acetone fixed cells to temperatures between 22°C and 100°C in 0.15 M NaCl, 0.015 M sodium citrate containing 4% formaldehyde for 20 min followed by cooling to room temperature and mounting in glycerol. Different cell types examined by the cytochemical method show markedly different heat denaturation curves. These differences appear to reflect differences in DNA-protein interaction in the deoxyribonucleoprotein complexes of nuclear chromatin. DNA in nuclei which are completely repressed and which have a tightly condensed chromatin, e.g. sperm heads and hen erythrocyte nucleim show greater stability to heat denaturation than does DNA in more active cell nuclei. The thermal stability of DNA in rat thymocyte nuclei, the most active cell nuclei examined, is as low as that of calf thymus DNA in solution. Hen erythrocyte nuclei show a two-step transition from the double-stranded to the single-stranded state. The first step occurs at a temperature which is slightly lower than that of isolated DNA, whereas the second step occurs at a higher temperature. Bull spermatozoa show a single-step transition and a T m value which is higher than that for bovine DNA. In all cell types examined, 85–100% of the DNA in the fixed cell nucleus appears to be in the double-stranded form.

Influence of netropsin and distamycin A on the secondary structure and template activity of DNA.

The action of netropsin and distamycin A on the DNA secondary structure and on the template properties of DNA in the DNA-and RNA-polymerase systems have been studied. Ultraviolet absorption measurements indicate characteristic hypochromic effects for the DNA-antibiotic complexes due to binding of netropsin and distamycin A to DNA. These are accompanied by the appearance of an absorption peak beyond 320 nm. This ultraviolet absorption maximum depends on the (A+T) content of DNA and disappears upon heat denaturation. These results together with earlier observations indicate an A and T specificity of the binding of the oligopeptides netropsin and distamycin A. As demonstrated by absorbance-temperature measurements netropsin increases considerably the thermal stability of DNA. Drastic changes can even be obtained with ratios below 5 moles netropsin per 100 DNA phosphate groups. The antibiotic dependent increase in the melting temperature is more pronounced for the DNA · netropsin than for the DNA · distamycin A complex. The structure of denatured DNA is also affected markedly by both antibiotics. From the results it is concluded that binding of netropsin and distamycin A to DNA is the result of their affinity to double-helical and base stacked regions. Comparison of the effect of these antibiotics on DNA-and RNA-synthesis in vitro shows that both netropsin and distamycin A inhibit the DNA- and RNA-polymerase reactions. In both systems the template activity of native as well as of denatured DNA is affected by these antibiotics. Using chromatin of Ehrlich ascites tumor cells as template it could be shown, that the nucleoprotein complex is as sensitive to the action of distamycin A as pure DNA.

Changes in deoxyribonucleoprotein during spermiogenesis in the bull: Sensitivity of DNA to heat denaturation

The thermal stability of DNA at various stages of spermiogenesis in bulls was studied by new cytochemical techniques. Fixed smears of testicular cells were heated to various temperatures in the presence of formaldehyde. After cooling, the extent of single-stranded DNA regions was measured by microspectrophotometric determination of the increment in UV-absorbancy at 265 nm or by microfluorimetry after acridine orange (AO) staining. The extent to which DNA had become denatured into a single-stranded state was determined from the magnitude of a shift in the fluorescence emission spectrum. Although UV-microspectrophotometry and acridine orange microfluorimetry measure different physical phenomena, the present results show that it is possible to follow the denaturation of DNA (“melting profile”) in intact cell nuclei by both these techniques. Since both techniques were used in parallel throughout the present investigation it has been possible to compare the two techniques relative to each other. The main biological conclusion from the present work is that during spermiogenesis the DNA becomes progressively more resistant to heat denaturation. This is thought to be due to changes in the state and composition of the deoxyribonucleoprotein. The relationship of these DNP changes to genome inactivation and chromatin condensation is briefly discussed.

Thermal stability of T2 DNA in situ

Thermal denaturation experiments were carried out on solutions of formaldehydefixed T2 bacteriophage and purified T2 DNA at low salt concentrations. Measurements of the flow birefringence, ultraviolet absorbance, and sedimentation velocity indicated that the tertiary structure of the internal DNA remains intact even at temperatures up to 20 ° higher than the melting temperature of free DNA under comparable conditions.

Polarographic investigation of binding of Cu++ and Cd++ by DNA.

AbstractThe equilibrium binding constants of Cd++ and Cu++ to native and denatured calf thymus DNA were determined polarographically. The binding constants are an exponential function of the potential at the binding site and as such they vary with ionic strength and with the charge on the DNA molecule. The correlation between the fraction of sites occupied by heavy metal ions and between the thermal stability of DNA in solution is discussed.