Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

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.     

Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis

A LightCycler real-time polymerase chain reaction (PCR) assay has been developed to detect and differentiate four of the main Leishmania species of the Old World. The assay is based on fluorescence melting curve analysis of PCR products generated from the minicircles of kinetoplast DNA. According to the melting temperature, which is a function of GC/AT ratio, length and nucleotide sequences of the amplified product, Leishmania major was differentiated from L. donovani and from L. tropica and L. infantum. Melting curves analysis offers a rapid alternative for identification of species in diagnostic or epidemiological studies of leishmaniasis or asymptomatic parasitism.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Calculation of melting curves for DNA

A method is reported for calculating the melting curve of a DNA molecule of random base sequence, including in the formalism the dependence of the free energy of base pair formation on the size of a denatured section. Some explicit results are shown for a “typical” base sequence, in particular the probability of helix formation at individual base pairs in several different regions of the molecule and the amount of melting from the end of the chain. Particular attention is drawn to the variation of local melting behavior from one region of the molecule to another. It is found that sections rich in AT melt at relatively low temperatures with a fairly broad transition curve, whereas regions rich in GC pairs melt at higher temperatures (as expected) with a very abrupt, local transition curve. To account qualitatively for the results one may divide melting into two kinds of processes: (a) the nucleation and growth of denatured regions, and (b) the merging together of two denatured sections at the expense of the intervening helix. The first of these processes dominates in the first stages of melting, and leads to rather broad local melting curves, whereas the second process predominates in the later stages, and occurs, in a particular part of the molecule, over a very narrow temperature range. It is estimated that the average length of a helix plus adjacent coil section at the midpoint of the transition is approximately 600 base pairs. Since transition curves which measure the local melting behavior reflect local compositions fluctuations, these curves contain information about the broad outlines of base sequence in the molecule. Some suggestions are made concerning experiments by which this potential information source could be exploited. In particular, it is pointed out that one might hope to map AT or GC rich regions at particular genetic loci in a biologically active DNA molecule. Values of the relevant parameters found earlier for the transition of homopolymers produce melting curves for a DNA of random base sequence which are in good agreement with the experimental transition curve for T2 phage DNA. Hence the present theoretical picture of the melting of polynucleotides is at least internally self-consistent.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

Isolation and Characterization of the Fertility Factor P of Vibrio cholerae

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.     

Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

Effect of DNA flanking sequence on charge transport in short DNA duplexes

Several factors can influence charge transport (CT)-mediated DNA, such as sequence, distance, base stacking, base pair mismatch, conformation, tether length, etc. However, the DNA context effect or how flanking sequences influence redox active drugs in the DNA CT reaction and later in DNA enzymatic repair and synthesis is still not well understood. The set of seven DNA molecules in this study have been characterized well for the study of flanking sequence effects. These DNA duplexes are formed from self-complementary strands and contain the common central four-base sequence 5'-A-G-C-T-3', flanked on both sides by either (AT)(n) or (AA)(n) (n = 2, 3, or 4) or AA(AT)(2). UV-vis, fluorescence, UV melting, circular dichroism, and cyclic voltammetry experiments were used to study the flanking sequence effect on CT-mediated DNA by using daunomycin or adriamycin cross-linked with these seven DNA molecules. Our results showed that charge transport was related to the flanking sequence, DNA melting free energy, and ionic strength. For (AA)(n) or (AT)(n) species of the same length, (AA)(n) series were more stable and more efficient CT was observed through the (AA)(n) series. The same trend was observed for (AA)(n)() and (AT)(n) series at different ionic strengths, further supporting the idea that flanking sequence can result in different base stacking and modulate charge transport through these seven DNA molecules.     

Physico-chemical determination of the ploidy of the unicellular alga, Chlorella pyrenoidosa (strain 211/8b) (author's transl)]

The ploidy of the unicellular green alga Chlorella pyrenoidosa (strain 211/8b) has been determined by means of renaturation kinetics. The nuclear DNA is made up from fast, intermediate and slow renaturing sequences, which represent respectively about 5, 15 and 80% of the DNA. These observations are consistent with the findings in other eukaryotic nuclear DNAs. Nevertheless, the relative importance of the repeated sequences is much lower than that observed in Chlamydomonas reinhardi [16] and in higher plants [18-20], but slightly higher than that obtained in Chlorella vulgaris [17]. The kinetic complexity of the main fraction of the Cl. pyrenoidosa nuclear DNA is found to be 2.94 - 10-10 daltons (mean value of five independant experiments) assuming value of 2.1 - 10-8 daltons for Cl. pyrenoidosa chloroplastic DNA. When compared with the analytical complexity of this fraction (80% of the nuclear DNA analytical complexity, that is 2.02 - 10-10 daltons), one can assume that the slow renaturing fraction of the nuclear DNA is constituted by a unique nucleotide sequence. This result thus suggests that Cl. pyrenoidosa (strain 211/8b) is an haploid organism. The possible existence of an haploid genome in the nuclei of the algae from Chlorella genus and the apparent absence of sexuality might explain the high discrepancy observed in the G + C content of the Chlorella nuclear DNAs.     

Mechanical stability of low-humidity single DNA molecules

DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone, which together with base sequence, further confer its higher order structure. The authors overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. The authors found that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70 and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules, which interact with DNA in vivo.     

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.     

Analysis of DNA of male and femaleSphaerocarpos donnellii Aust. (Liverwort)

Summary The karyotypes of females and males ofSphaerocarpos donnellii differ in that there is a large essentially heterochromatic X chromosome in the female (approx. 25 volume-% of the autosomes) and a small Y chromosome in the male (0.1–3 volume-% of the autosomes). DNA from females and males differ in buoyant densities in cesium chloride equilibrium gradients (1.702₅ and 1.703₅g cm^-3, respectively) and in melting points (87.5 and 88.5°C in SSC). The differences are statistically significant. Base analyses revealed 2.5 Mol-% of the rare base 5-methyl cytosine. Upon reassociationSphaerocarpos DNA behaves kinetically in a heterogeneous manner. About 22% of the DNA is repetitive with an average kinetic complexity of 1.6×10⁸ daltons. The kinetic complexity of the slow reassociating DNA fraction, considered to be of the single copy type, is 3.2×10¹⁰ daltons. No difference in the renaturation behavior between DNA of males and females could be detected with the techniques used. Our data thus indicate that X chromosomal DNA cannot contain large amounts of highly repeated nucleotide sequences and that it is slightly enriched in AT content compared to the autosomes.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.     

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.     

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process.

The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.     

High diversity in DNA of soil bacteria.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)