High resolution thermal denaturation of mammalian DNAs.

High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed.     

Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm³, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm³ appear to be heterogeneous.     

The use of netropsin with CsCl gradients for the analysis of DNA and its application to restriction nuclease fragments of ribosomal DNA from Physarum polycephalum

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, deltarho, at saturation is given by deltarho = -109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA + dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two-site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarum polycephalum nucleolar satellite DNA from main-band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non-transcribed sequences in this DNA have similar nucleotide compositions.     

The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid.

The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other. Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites. Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs. The binding of actinomycin to DNA was decreased by the presence of netropsin. Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding. Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain. Supercoiled DNA gave the same result as linear DNA. These results imply that DNA can absorb alterations in conformation within a short distance.     

Cat satellite DNA. Isolation using netropsin with CsCl gradients

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.     

Organization of nucleotide sequences in the chicken genome

The four major components of chicken DNA were prepared by density gradient centrifugation and characterized in several basic properties: relative amounts, dG + dC content, buoyant densities, compositional heterogeneity, and reassociation kinetics. While the relative amounts and the compositions of the major components of chicken DNA were similar to those found in mammalian genomes, their compositional heterogeneities were found to be narrower. The relative amounts of interspersed repeated and unique sequences were strikingly different in different components and also different from those found in the corresponding major components of mouse and human DNAs. If one takes into consideration that major DNA components (a) account for practically all of main-band DNA and (b) derive by preparative breakage from very long DNA segments of fairly homogeneous composition, the isochores, our findings indicate that the distribution of interspersed repeats is different in different chromosomal regions and is species-specific.     

Base Composition of the DNA of Mycoplasma-Like Organisms Associated with Various Plant Diseases

DNA was isolated from periwinkle plants infected by various mycoplasma-like organisms (MLOs), and from apple trees affected by apple proliferation. The DNA of the causal agents was separated from the host plant DNA by repeated bisbenzimide-CsCl buoyant density gradient centrifugation which resulted in highly enriched MLO DNA bands characterized by a lower buoyant density than that of the host DNA. The MLO DNAs were hydrolyzed to free bases which were determined by HPLC. The analyses revealed a similar low G + C content as found in the DNAs of several culturable mycoplasmas and spiroplasmas. The values of the DNA of the agents of the diseases investigated were as follows: European aster yellows 23.0, periwinkle virescence 23.5, apple proliferation 23.7, rape virescence 24.2, and phyllody of Diplotaxis erucoidcs 26.2 mol % G + C, respectively. Methylated bases were detected in low amounts only.     

Liverwort genomes display extensive structural variations

PIKE, L. M., HU, A., RENZAGLIA, K. S. & MUSICH, P. R., 1992. Liverwort genomes display extensive structural variations. Analyses of the total genomic DNA of eight species of liverworts and two species of green algae by thermal denaturation and CsCl buoyant density gradient centrifugation reveal a high degree of structural complexity and interspecific heterogeneity. The hepatic taxa exhibit two or more DNA components of varying base composition. Average G4-C contents of total cellular DNA calculated from melting profiles are similarly variable, ranging from 38% to 53% G + C. The green alga Chara , a member of the ancestral line to land plants, shows similarities with liverworts in possessing multiple DNA components of comparable complexity, whereas Hydrodiciyon DNA displays a single component. Detailed hybridization analyses of individual density gradient fractions using α-tubulin, rRNA and ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene probes were performed to locate the low-copy number and moderately repetitive nuclear genes, and the chloroplast chromosome, respectively. The location of each gene within the density gradient is highly variable among the organisms examined; a-tubulin occurs in fractions ranging from 44–64% G + C, rDNA in 50–64% G + C fractions, and the RbcL gene is located in fractions from 30–59% G + C. For a given species, the two nuclear genes normally overlap in their distributions within the gradient. In most instances, neither gene occurs in the major DNA components, indicating that these components may contain repetitive DNAs. The observed variation in the density of the rbcL gene implies substantial reorganization of the chloroplast genome. The overall differences in the genomic components within and between taxa provide insight into the dynamics of DNA structure that have occurred during the extended evolutionary history of these organisms.     

Effect of ligand binding on the buoyant density of DNA in Nycodenz gradients.

The effect of ligand binding upon the buoyant density of DNA in Nycodenz gradients has been studied using DNAs of differing base compositions. The effect of both intercalating ligands (ethidium bromide and proflavin) and non-intercalating ligands (distamycin A, DAPI and netropsin) has been studied. The binding of intercalating ligands to DNA has essentially no effect on the buoyant density of DNA in Nycodenz gradients. The non-intercalating ligands were found to increase the buoyant density of DNA in a base specific manner. The increase in buoyant density can be interpreted in terms of disruption of the hydration shell of the DNA molecule caused by the binding of the ligand along the minor groove of the DNA helix.     

Interaction of nucleic acids with a non-intercalative anti-leukemic compound containing bisquarternary heterocycles

The binding of an antitumour drug with bisquarternary ammonium heterocyclic structure, NSC-101327, to nucleic acids has been examined by using ultraviolet absorption and CD measurements. Like the minor groove-binding oligopeptides, netropsin and distamycin A, the optically inactive chromophoric system of NSC-101327 shows induced Cotton effects in the CD spectra of complexes with various DNAs, RNA and single-stranded polynucleotides. This property directly reflects interaction of NSC-101327 with different types of nucleic acids at moderate ionic strength, which contrasts with previous findings of a higher selective binding of netropsin to B-DNA. However, an efficient interactin of NSC-101327 with dA·dT basepair sequences is demonstrated by a large melting temperature increase of dA·dT-rich DNAs. NSC-101327 also reacts with dG·dC base pairs of B-DNA and forms a complex with Z-DNA of poly(br⁸dG-dC)·poly(br⁸DG-dC). The affinity of NSC-101327 to poly(dG-dC)·poly(dG-dC) is, however, lower, and the CD spectral binding effect depends on the ionic strength. The CD results of the complex with poly(dA-dT)·poly(dA-dT) suggests at least two binding modes, in accordance with previous conclusions. This is indicated by a clear-cut initial increase of the CD signal and a subsequent large decrease to negative CD signals. Competition experiments with netropsin suggest that binding of NSC-101327 occurs preferentially in the minor groove without intercalation. NSC-101327 also tends to interact with lower binding affinity to dG-dC pairs in B-DNA, with rA·rU pairs of RNA and with single-stranded polynucleotides. Thus our results suggest that NSC-101327 represents a DNA groove-binding ligand of lower basepair specificity and lower conformational selectivity compared to the B-specific netropsin probe.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Complexity in the binding of minor groove agents: netropsin has two thermodynamically different DNA binding modes at a single site

Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect.     

Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

Related base sequences in the DNA of simple and complex organisms. VI. The extent of base sequence divergence among the DNAs of various rodents

The magnitude of differences which occur in the base sequence of DNAs from related animals was investigated through studies of four rodent DNAs. Fractionation on preparative CsCl density gradients and detailed examination of thermal denaturation profiles revealed the existence of several distinguishable components comprising the main band in addition to satellite bands. No difference was apparent in the distribution of unique and partially redundant sequences among these main band components, or in the apparent rate at which base substitution has occurred in evolution. Comparisons of sequence were also made by annealing reactions in solution at high concentration for extended periods with either labeled total mouse or rat DNA, or labeled unique sequences of mouse DNA. These experiments permitted assessment of the absolute extent of cross-reaction, as well as the extent of sequence divergence which had occurred between the two groups of interacting DNA molecules. Some implications of the apparent rapidity of nucleotide substitution in DNA are discussed.This research was supported by a grant from the National Science Foundation (GB 6099).     

DNA and the theory of stabilizing selection

A comparison of structural-functional features of genomic DNAs allowed to estimate the role of internal and external factors in evolution of different groups of organisms. The basic difference between higher and lower organisms has been demonstrated. It is reflected in the difference of their reaction on to external factors in accordance with two adaptation types, the openness and autonomization. There is a correlation between structural-functional organization of genomic DNAs of higher and lower organisms and the above mentioned types of adaptation. DNA of lower organisms has been proposed to be characterized as "labile", and that of higher organisms, as "stable". The "DNA lability" means high mutation ability, which characterizes the existence of and evolution of lower organisms (genetic inconstancy of the lower organisms). On the contrary, "DNA stability" means the creation of stable genetic apparatus, reduction of variability in higher organisms (genetic constancy of higher organisms). This suggests the existence of the two principal ways of evolution.     

Use of preparative CsCl gradients to estimate DNA buoyant densities: potential errors due to fractionation artifacts

The equilibrium banding behavior of high molecular weight DNA has been examined after centrifugation in CsCl gradients on angle rotors in the Spinco preparative ultracentrifuge. DNAs of base composition ranging from 30% GC to 71% GC, from a variety of species, band in close agreement to a calculated curve relating density and radial distance in angle preparative gradients, but only if the DNA is sheared prior to centrifugation. Unsheared DNA, after bottom tube puneture and drop collection by conventional methods, displays a characteristic unsymmetrical profile and the fraction with the largest concentration of DNA appears in a much lower fraction number than expected. This artifact leads to misealculation of buoyant densities from preparative gradients and interferes with fractionation of DNA, as for instance in separating satellite bands. Collection of the gradient from the top reverses the direction of the profile asymmetry and shearing eliminates it entirely.     

Procaryotic genomic DNA inhibits mammalian cell transformation.

Ltk- mouse cells were transformed to thymidine kinase prototrophy in the presence of carrier DNAs isolated from different organisms. Procaryotic genomic and phage DNA was consistently less effective as a carrier than was eucaryotic DNA. Mixing experiments indicate that DNA of procaryotic origin inhibits mammalian cell transformation.     

Compositional transitions in the nuclear genomes of cold-blooded vertebrates

Summary The compositional properties of DNAs from 122 species of fishes and from 18 other coldblooded vertebrates (amphibians and reptiles) were compared with those from 10 warm-blooded vertebrates (mammals and birds) and found to be substantially different. Indeed, DNAs from cold-blooded vertebrates are characterized by much lower intermolecular compositional heterogeneities and CsCl band asymmetries, by a much wider spectrum of modal buoyant densities in CsCl, by generally lower amounts of satellites, as well as by the fact that in no case do buoyant densities reach the high values found in the GC-richest components of DNAs from warm-blooded vertebrates.In the case of fish genomes, which were more extensively studied, different orders were generally characterized by modal buoyant densities that were different in average values as well as in their ranges. In contrast, different families within any given order were more often characterized by narrow ranges of modal buoyant densities, and no difference in modal buoyant density was found within any single genus (except for the genusAphyosemion, which should be split into several genera).The compositional differences that were found among species belonging to different orders and to different families within the same order are indicative of compositional transitions, which were shown to be essentially due to directional base substitutions. These transitions were found to be independent of geological time. Moreover, the rates of directional base substitutions were found to be very variable and to reach, in some cases, extremely high values, that were even higher than those of silent substitutions in primates. The taxonomic and evolutionary implications of these findings are discussed.     

Effect of salts and chromatin concentrations on the buoyant density of chromatin in metrizamide gradient.

Using isopycnic centrifugation in metrizamide gradient, effect of ions and chromatin concentration on the buoyant density of chromatin was quantitatively examined. An elevation followed by gradual decline and secondary increase of the density occurred in accordance with increase in MgCl2 or NaCl concentration. Maximum density was observed at a concentration of these salts known to result in the condensation of chromatin. Release of protein occurred during the phase of density decline. The second increase in density is mainly due to the density increment of DNA in the chromatin. The density was dependent upon the concentration of chromatin in a band formed in the metrizamide gradient, while the density of free DNA and protein was not so greatly affected by their concentration. The density of chromatin in the presence of 0.14 M NaCl was less affected by the chromatin concentration than that in the absence of salt. Calculation of results indicates that grade of hydration of chromatin at concentrations lower than 400 microgram/ml in 1 mM Tris-HCl (pH 8.0) is higher than that expected from its DNA and protein components.