Evidence for random distribution of sequence variants in Tenebrio molitor satellite DNA.

Tenebrio molitor satellite DNA has been analysed in order to study sequential organization of tandemly repeated monomers, i.e. to see whether different monomer variants are distributed randomly over the whole satellite, or clustered locally. Analysed sequence variants are products of single base substitutions in a consensus satellite sequence, producing additional restriction sites. The ladder of satellite multimers obtained after digestion with restriction enzymes was compared with theoretical calculations and revealed the distribution pattern of particular monomer variants within the satellite. A defined higher order repeating structure, indicating the existence of satellite subfamilies, could not be observed. Our results show that some sequence variants are very abundant, being present in nearly 50% of the monomers, while others are very rare (0-1% of monomers). However, the distribution of either very frequent, or very rare sequence variants in T. molitor satellite DNA is always random. Monomer variants are randomly distributed in the total satellite DNA and thus spread across all chromosomes, indicating a relatively high rate of sequence homogenization among different chromosomes. Such a distribution of monomer variants represents a transient stage in the process of sequence homogenization, indicating the high rate of spreading in comparison with the rate of sequence variant amplification.     

Detection of satellite DNA in Palorus ratzeburgii: analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA.

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satellite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

The structure of kinetoplast DNA. I. Properties of the intact multi-circular complex from Crithidia luciliae.

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution.

We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of sigma= -0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA superhelix were analysed. Evaluation of the whole plasmid yielded average superhelix dimensions of 27 +/- 9 nm (outer superhelix diameter D), 107 +/- 51 nm (superhelix pitch P), and 54 +/-8 degrees (superhelix pitch angle alpha). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 +/- 3.3 nm, P = 42 +/- 13 nm and alpha= 63 +/- 20 degrees for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface.     

Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome.

The pericentromeric region of the human X chromosome is characterized by a tandemly repeated family of 2.0 kilobasepair (kb) DNA fragments, initially revealed by cleavage of human DNA with the restriction enzyme BamHI. We report here the complete nucleotide sequence of a cloned member of the repeat family and establish that this X-linked DNA family consists entirely of alpha satellite DNA. Our data indicate that the 2.0 kb repeat consists of twelve alpha satellite monomers arranged in imperfect, direct repeats. Each of the alpha X monomers is approximately 171 basepairs (bp) in length and is 60-75% identical in sequence to previously described primate alpha satellite DNAs. The twelve alpha X monomers are 65-85% identical in sequence to each other and are organized as two adjacent, related blocks of five monomers, plus an additional two monomers also related to monomers within the pentamer blocks. Partial nucleotide sequence of a second, independent copy of the 2.0 kb BamHI fragment established that the 2.0 kb repeat is, in fact, the unit of amplification on the X. Comparison of the sequences of the twelve alpha X monomers allowed derivation of a 171 bp consensus sequence for alpha satellite DNA on the human X chromosome. These sequence data, combined with the results of filter hybridization experiments of total human DNA and X chromosome DNA, using subregions within the 2.0 kb repeat as probes, provide strong support for the hypothesis that individual human chromosomes are characterized by different alpha satellite families, defined both by restriction enzyme periodicity and by chromosome-specific primary sequence.     

Distribution and sequence homogeneity of an abundant satellite DNA in the beetle, Tenebrio molitor.

The mealworm beetle, Tenebrio molitor, contains an unusually abundant and homogeneous satellite DNA which constitutes up to 60% of its genome. The satellite DNA is shown to be present in all of the chromosomes by in situ hybridization. 18 dimers of the repeat unit were cloned and sequenced. The consensus sequence is 142 nt long and lacks any internal repeat structure. Monomers of the sequence are very similar, showing on average a 2% divergence from the calculated consensus. Variant nucleotides are scattered randomly throughout the sequence although some variants are more common than others. Neighboring repeat units are no more alike than randomly chosen ones. The results suggest that some mechanism, perhaps gene conversion, is acting to maintain the homogeneity of the satellite DNA despite its abundance and distribution on all of the chromosomes.     

Higher-Order Organization of Subrepeats and the Evolution of Cervid Satellite I DNA

Based on sequence analyses of 17 complete centromeric DNA monomers from ten different deer species, a model is proposed for the genesis, evolution, and genomic organization of cervid satellite I DNA. All cervid satellite I DNA arose from the initial amplification of a 31-bp DNA sequence. These 31-bp subrepeats were organized in a hierarchical fashion as 0.8-kb monomers in plesiometacarpalia deer and 1-kb monomers in telemetacarpalia deer. The higher-order repeat nature of cervid centromeric satellite DNA monomers accounts for their high intragenomic and intraspecific sequence conservation. Such high intraspecific sequence conservation validates the use of a single cervid satellite I DNA monomer from each deer species for interspecific sequence comparisons to elucidate phylogenetic relationships. Also, a specific 0.18-kb tandem duplication was observed in all 1-kb monomers, implying that 1-kb cervid satellite I DNA monomers arose from an unequal crossover event between two similar 0.8-kb ancestral DNA sequences. Received: 28 May 1996 / Accepted: 24 October 1996     

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.     

Compaction of single molecules of supercoiled DNA immobilized on amino mica: from duplex to minitoroidal and spheroidal conformations

Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica.     

Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be -0.5 degrees per base pair (bp). This is equivalent to a change of -0.15 +/- 0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55 +/- 0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38 +/- 0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained .     

Theoretical model for the equilibrium behavior of DNA superhelices

A statistical-mechanical model for superhelical DNA is presented. The partition function for a DNA superhelix is written by using a combinatorial approach in order to allow for the known relation between the number of superhelical twists and the states of the base pairs in the double helix. While the theory allows any factors which might contribute to the free energy of superhelical twisting to be included in the statistical weights of the superhelical twists, only the reduction in configurational entropy is considered in this paper. Similarities between an imperfectly matched DNA double helix and a DNA superhelix are used in the derivation of expressions for the entropy of superhelical DNA. Although the partition function is presented in a general form, permitting many equilibrium properties of DNA superhelices to be treated, the application considered in this paper is the calculation of helix–coil transition curves. Several experimentally observed features of such transitions are predicted. For example, the curves are bimodal, with an early and a late transition relative to that of a nicked molecule. The results are very sensitive to the volume within which two parts of the double helix must meet when forming a superhelical twist. The free energy of superhelix formation is calculated, and the results are compared with those obtained from the data of Bauer and Vinograd for ethidium bromide intercalation. In the present model, the free energy increases less sharply with an increase in the number of superhelical twists than observed experimentally, indicating that factors other than configurational entropy probably make important contributions to the free energy of superhelix formation.     

Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be −0.5° per base pair (bp). This is equivalent to a change of −0.15±0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55±0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38±0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained.     

Structure of plectonemically supercoiled DNA

Using electron microscopy and topological methods, we have deduced an average structure for negatively supercoiled circular DNA in solution. Our data suggest that DNA has a branched plectonemic (interwound) form over the range of supercoiling tested. The length of the superhelix axis is constant at 41% of the DNA length, whereas the superhelix radius decreases essentially hyperbolically as supercoiling increases. The number of supercoils is 89% of the linking deficit. Both writhe and twist change with supercoiling, but the ratio of the change in writhe to the change in twist is fixed at 2.6:1. The extent of branching of the superhelix axis is proportional to the length of the plasmid, but is insensitive to superhelix density. The relationship between DNA flexibility constants for twisting and bending calculated using our structural data is similar to that deduced from previous studies. The extended thin form of plectonemically supercoiled DNA offers little compaction for cellular packaging, but promotes interaction between cis-acting sequence elements that may be distant in primary structure. We discuss additional biological implications of our structural data.     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

A new AluI satellite DNA in the root-knot nematode Meloidogyne fallax: relationships with satellites from the sympatric species M. hapla and M. chitwoodi

A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax (Nematoda, Tylenchida) genome was cloned and sequenced. The satellite monomer is 173 bp long and has a high A + T content of 72.3%, with frequent runs of A's and T's. The sequence variability of the monomers is 2.7%, mainly due to random distribution of single-point mutations. A search for evidence of internal repeated subunits in the monomer sequence revealed a 6-bp motif (AAATTT) for which five degenerated repeats, differing by just a single base pair, could be identified. Pairwise comparison of the M. fallax satellite with those from the sympatric species Meloidogyne chitwoodi and Meloidogyne hapla revealed a high sequence similarity (68.39%) with one satellite DNA subfamily in M. chitwoodi, which indicated an unexpected close relationship between them. Given the high copy number and the extreme sequence homogeneity among monomeric units, it may be assumed that the satellite DNA of M. fallax could have evolved through some recent and extensive amplification burst in the nematode genome. In this case, its relatively short life would not yet have allowed the accumulation of random mutations in independent amplified repeats. Considering the morphological resemblance between the two species and their ability to produce interspecific fertile hybrids under controlled conditions, these results indicate that M. fallax may share a common ancestor with M. chitwoodi, from which it could have diverged recently. All these data suggest that M. fallax could be the result of a recent speciation process and show that Meloidogyne satellite DNAs may be of interest to resolve phylogenetic relationships among closely related species from this genus.      

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.