Structural property of DNA that migrates faster in gel electrophoresis, as deduced by CD spectroscopy

Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form-like structure has been suggested. Reduction in DNA length due to the formation of the A-form-like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.     

Satellite DNA and chromosomes in Neotropical fishes: methods,applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C₀t–1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII‐DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.     

Suppression of electrophoretic anomaly of bent DNA segments by the structural property that causes rapid migration.

Intrinsic DNA curvature is speculated to be a common feature of all satellite DNA sequences and may aid in the tight winding of DNA in constitutive heterochromatin. Several satellite DNAs, however, show unusually rapid migration in non-denaturing polyacrylamide gels, which is just the opposite behavior of that shown by curved DNA structures. Employing bovine satellite I DNA monomer, we attempted to understand the molecular mechanism of 'rapid migration'. The phenomenon of rapid migration was temperature-dependent and to a small extent polyacrylamide-concentration-dependent. Physiological or near-physiological concentrations of Mg2+and Ca2+ions bent the rapid migrating DNA segment. Predominance of purine-purine base stacking over purine-pyrimidine in nucleotide sequence was strongly indicated to be the cause of the rapid migration. Furthermore, they seemed to be implicated in the formation of induced DNA bend. We also found that the satellite I monomer contains an intrinsic DNA curvature as do many other satellites. Heretofore, the rapid migration property has concealed the presence of curvature.     

Conformations Consistent with Charge Migration Observed in DNA and RNA X-ray Structures

Abstract

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.     

A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.     

Primary moult of continental Black-tailed Godwits Limosa limosa limosa in the Doñana wetlands,Spain

Capsule: Most Continental Black-tailed Godwits Limosa limosa limosa using the Doñana wetlands during post-breeding migration appear to begin moult before they arrive and suspend moult before they migrate onwards to West Africa.

Aims: We aim to describe the primary moult strategies and patterns in the Continental Black-tailed Godwits using the Doñana wetlands, a major passage and wintering area for waterbirds in southern Spain.

Methods: Individual godwits were captured, marked and their primary moult was scored in Doñana during the non-breeding season (June–March) in 2011 and 2012. Data from resightings of colour-marked godwits and birds equipped with satellite transmitters were used to estimate stopover duration during post-breeding migration (June–September) to determine if godwits move to West Africa before completing their primary moult.

Results: Average primary moult duration was estimated to be 84 days?±?9 se, during 29 June–21 September and did not differ between sexes. Only 2% of individuals were observed with suspended moult. We estimated stopover duration in Doñana to be 13 days?±?2 se before migrating to West Africa.

Conclusions: Most godwits stage for about two weeks in the Doñana wetlands during southward migration, moult their primaries and appear to suspend moult before crossing the Sahara. Others may complete their primary moult in Doñana, or elsewhere in Europe and overwinter in Doñana where increasing numbers of godwits have been detected in recent years. A few individuals may finish the moult in Doñana and migrate to West Africa late in the post-breeding season.     

Evolution of Satellite DNAs in a Radiation of Endemic Hawaiian Spiders: Does Concerted Evolution of Highly Repetitive Sequences Reflect Evolutionary History?

Satellite DNAs are known for an unusual and nonuniform evolution characterized by rapid evolutionary change between species and concerted evolution leading to molecular homogeneity within species. In this paper we use satellite DNAs for phylogenetic analysis of a rapidly evolving lineage of spiders and compare the phylogeny with a hypothesis previously generated based on mitochondrial DNA and allozymes. The spiders examined include almost all species within a monophyletic clade of endemic Hawaiian Tetragnatha species, the spiny-leg clade. The phylogeny based on satellite sequences is largely congruent to those produced by mtDNA and allozymes, except that the satellite DNA yields much longer branches, with higher levels of support for any given node. Closely related species that have differentiated ecologically within an island are well resolved with satellite DNA but much less so with mtDNA. These results suggest that Tetragnatha stDNA repeats seem to be evolving gradually and cohesively during the diversification of these endemic Hawaiian spiders. The study also reveals gain–loss of satellite DNA copies during species diversification. We conclude that satellite DNA sequences may potentially be very useful for resolving relationships between rapidly evolving taxa within an adaptive radiation. In addition, satellite DNA as a nuclear marker suggests that hybridization or peripatry could play a possible role in species formation that cannot be revealed by mitochondrial markers due to its maternal inheritance.This article contains online supplementary data.Reviewing Editor: Dr. Rafael Zardoya     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

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.     

A short GC‐rich palindrome of human mannose receptor gene coding region displays a conformational switch

Conformational switching in DNA is fundamental to biological processes. The structural status of a palindromic GC‐rich dodecamer DNA sequence, integral part of human MRC2 coding region, and a related sequence of opposite polarity from human FDX1 gene were characterized and compared. UV‐melting, circular dichroism, and gel electrophoresis experiments demonstrated the formation of intermolecular structures. Although stability and molecularity of both the oligomeric structures were found to be almost identical, their secondary structures differed remarkably as A1 MRC2 sequence showed A‐like and B‐like DNA conformation, whereas the A2 FDX1 sequence exhibited only the A‐like signatures. The study is relevant for understanding structural polymorphism at genomic locations depending on DNA sequence and solution environment. © 2012 Wiley Periodicals, Inc. Biopolymers 97:950–962, 2012.     

Deoxyribonucleic Acid Base Composition of Some Cellular Slime Molds*

SYNOPSIS. Deoxyribonucleic acids (DNAs) isolated from 12 representatives of cellular slime molds grown in monoxenic cultures were analyzed by density gradient centrifugation in CsC1. The unique contribution of the DNA of each food organism was subtracted and the DNA base composition of each cellular slime mold determined. The guanine plus cytosine contents range 22–37 moles % within the representatives of the order. Minor bands (satellite bands) of DNA have been observed in preparations from Dictyostelium spp. and from Polysphondylium pallidum.     

蓝狐-卫星DNA的克隆及序列分析

利用限制性内切酶酶切蓝狐基因组, 经琼脂糖凝胶电泳, 对特异性亮带进行克隆、测序及序列分析。结果获得42个卫星DNA序列, 该卫星DNA单体大小为737 bp, G+C含量为51.9%, 单体之间同源性为91%~97%; 每个单体由3个约245 bp的亚重复串联构成, 亚重复之间的同源性为49%~55%; 在物种进化过程中, 该卫星DNA有G+C含量逐渐降低而A+T含量逐渐上升的趋势; 该卫星DNA为犬科动物种属所特有, 与犬着丝粒相关卫星DNA为同类卫星DNA, 同源性为74%, 命名为α-卫星DNA。     

The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

The Affinity of DNA-Microtubule Protein Complexes and their Disruption by Tubulin Binding Drugs

Abstract

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar K_app values in the range of 2.1×10⁷ M^?1 to 2.0×10⁸ M^?1. CEN11 DNA-MTP complexes had by far the highest K_app value of 2.0×10⁸ M^?1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites -(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher K_app value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10–100 fold higher drug concentrations to disrupt the CEN 11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383–392,1984; Marx and Denial, Molecular Basis of Cancer 172B,65-15 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

Nucleic acid aggregation geometry and the possible evolutionary origin of ribosomes and the genetic code

We probed the structure of mammalian repetitive DNAs with a site-specific mammalian endodeoxyribonuclease, which we recently identified, and which apparently represents a common enzyme activity among the mammals (McKenna et al., 1981). With several of the DNAs (e.g. mouse satellite, guinea pig β-satellite, variable repeated spacer DNA from mouse ribosomal genes and primate alphoid sequences), the endonuclease activity gave highly specific cleavage patterns when the digestion products were analyzed by gel electrophoresis. These patterns were not always identical to those produced by microbial restriction enzymes. However, in other cases (e.g. bovid and caprid satellites and guinea pig α-satellite) the repetitive DNAs appeared to be degraded randomly. Thus, the mammalian enzyme reveals structural features of the repetitive sequences that are not rendered immediately obvious by microbial restriction enzyme analysis. Evidence from mapping data presented here suggests that the mammalian site-specific endonucleases are not sequence specific but have special affinity for imperfect or hyphenated palindromic sequences in repetitive DNAs and in other eukaryotic DNA sequences.     

Molecular mechanics calculations of dA12·dT12 and of the curved molecule d(GCTCGAAAAA)4·d(TTTTTCGAGC)4

Using the AMBER software package (Weiner and Kollman 1981) substantially modified for electrostatic contributions, the structural energies of the double-stranded oligonucleotides dA₁₂·dT₁₂ and d(GCTCGAAAAA)₄·d(TTTTTCGAGC)₄ were minimized. Using various starting structures for the molecule dA₁₂·dT₁₂, one final structure is obtained which possesses the experimentally determined properties of poly(dA)·poly(dT). This structure is an A-form-B-form-hybrid structure similar to that of Arnott et al. (1983). The dA-strand is similar to an A-form while the dT-strand is similar to normal B-form. This structure and separately optimized B-form sequence stretches were used to construct the double-stranded fragment d(GCTCGAAAAA)₄ which again was optimized. This sequence, when imbedded in a DNA fragment as contiguous repeats, shows a gel migration anomaly which has been interpreted as stable curvature of the DNA (Diekmann 1986). The calculated structure of this sequence indeed has a curved helix axis and is discussed as a model for curved DNA. A theoretical formalism is presented which allows one to calculate the structural parameters of any nucleic acid double helix in two different geometrical representations. This formalism is used to determine the parameters of the base-pair orientations of the curved structure in terms of wedge as well as cylindrical parameters. In the structural model presented here, the curvature of the helix axis results from an alternation of two different DNA structures in which the base-pairs possess different angles with the helix axis (cylinder tilt). Resulting from geometric restraints, a negative cylinder tilt angle correlates strongly with the closing of the minor groove (wedge roll). The blocks with different structure are not exactly coincident with the dA₅-blocks and the B-DNA stretches. Within the dA₅ block, base-pair tilt and wedge roll adopt large values which proceed into the 3 flanking B-DNA sequence by about one base-pair. These properties of the structure calculated here are discussed in terms of different models explaining DNA curvature.     

Differential homogenization and amplification of two satellite DNAs in the genus Cucurbita (Cucurbitaceae)

Two different satellite DNAs exist in the genus Cucurbita which are different with respect to repeat length (350 by and 170 bp), array size, and sequence homogenization. Whereas the 350-bp satellite DNA is prominent and very homogeneous in all species investigated except for C. maxima and C. lundelliana, the 170-bp satellite is rather evenly distributed in all species. In C. maxima and C. lundelliana the 350-bp satellite is present only in small amounts, but detectable by the sensitive PCR method. These repeats are also very homogeneous, reflecting a silent stage of satellite DNA. In contrast, the 170-bp satellite DNA is intra- and interspecifically heterogeneous. It is striking that the species with no detectable amount of 350-bp satellite contain 170-bp satellite DNA clusters with the highest degree of homogeneity. The evolution of satellite DNA repeats within cultivated and wild species in the genus Cucurbita is elucidated using the sequence data of both satellite DNAs from all species investigated. The value of satellite DNA for phylogenetic analysis between closely related species is discussed. Correspondence to: V. Hemleben     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

The influence of CpG (5′-d(CpG)-3′ dinucleotides) methylation on ultrasonic DNA fragmentation

Abstract

DNA methylation is an important way of gene regulation. The variety of methods for DNA methylation analysis based on chemical modification or enzyme digestion has been proposed. However, DNA is able to undergo transformations under physical power. Here, we report that the cytosine methylation in CpG dinucleotides determines the difference in fragmentation rate of methylated and unmethylated DNA under sonication. We found that at the beginning of sonication, methylated DNAs are degraded faster than unmethylated one, and the difference in fragmentation degree can be evaluated with high reliability by quantitative polymerase chain reaction (qPCR). The optimal parameters that provide the greatest difference in amount of amplifiable DNA targets corresponding to fragmentation degree are the following: moderate amplicon size (about 150–250?bp), medium CpG sparseness (one CpG dinucleotide per ～12–14 nucleotides of the chain), and short sonication time (less than 5?min). Along with CpG, the CpA and CpT contents of amplified regions should be taken into account for proper DNA fragmentation by ultrasound as well. The obtained data could be used for elaboration of a method for comparative methylation testing, when there is no need to detect methylation of certain CpG dinucleotides. This method will be simple (can be used by any technician familiar with PCR), low cost (no need to use an expensive reagents), and fast (only brief DNA sonication and conventional qPCR are carried out).

Communicated by Ramaswamy H. Sarma