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Background: Ubiquitous presence of short extrachromosomal circular DNAs (eccDNAs) in eukaryotic cells has perplexed generations of biologists. Their widespread origins in the genome lacking apparent specificity led some studies to conclude their formation as random or near-random. Despite this, the search for specific formation of short eccDNA continues with a recent surge of interest in biomarker development., Results: To shed new light on the conflicting views on short eccDNAs' randomness, here we present DeepCircle, a bioinformatics framework incorporating convolution- and attention-based neural networks to assess their predictability. Short human eccDNAs from different datasets indeed have low similarity in genomic locations, but DeepCircle successfully learned shared DNA sequence features to make accurate cross-datasets predictions (accuracy: convolution-based models: 79.65 ± 4.7%, attention-based models: 83.31 ± 4.18%)., Conclusions: The excellent performance of our models shows that the intrinsic predictability of eccDNAs is encoded in the sequences across tissue origins. Our work demonstrates how the perceived lack of specificity in genomics data can be re-assessed by deep learning models to uncover unexpected similarity., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Extrachromosomal circular DNA (eccDNA) is a kind of DNA that widely exists in eukaryotic cells. Studies in recent years have shown that eccDNA is often enriched during tumors and aging, and participates in the development of cell physiological activities in a special way, so people have paid more and more attention to the eccDNA, and it has also become a critical new topic in modern biological research., Description: We built a database to collect eccDNA, including animals, plants and fungi, and provide researchers with an eccDNA retrieval platform. The collected eccDNAs were processed in a uniform format and classified according to the species to which it belongs and the chromosome of the source. Each eccDNA record contained sequence length, start and end sites on the corresponding chromosome, order of the bases, genomic elements such as genes and transposons, and other information in the respective sequencing experiment. All the data were stored into the TeCD (The eccDNA Collection Database) and the BLAST (Basic Local Alignment Search Tool) sequence alignment function was also added into the database for analyzing the potential eccDNA sequences., Conclusion: We built TeCD, a platform for users to search and obtain eccDNA data, and analyzed the possible potential functions of eccDNA. These findings may provide a basis and direction for researchers to further explore the biological significance of eccDNA in the future., (© 2023. The Author(s).)

Extrachromosomal circular DNA (eccDNA) is a double-stranded DNA molecule found in various organisms, including humans. In the past few decades, the research on eccDNA has mainly focused on cancers and their associated diseases. Advancements in modern omics technologies have reinvigorated research on eccDNA and shed light on the role of these molecules in a range of diseases and normal cell phenotypes. In this review, we first summarize the formation of eccDNA and its modes of action in eukaryotic cells. We then outline eccDNA as a disease biomarker and reveal its regulatory mechanism. We finally discuss the future prospects of eccDNA, including basic research and clinical application. Thus, with the deepening of understanding and exploration of eccDNAs, they hold great promise in future biomedical research and clinical translational application., Competing Interests: YZ, LY, SZ, XS, XZ No competing interests declared, (© 2022, Zhao et al.)

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal library.

We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed laser-triggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved., (© 2015 Society for Laboratory Automation and Screening.)

Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis.

Double-strand DNA breaks (DSBs) resulting from cellular metabolic processes and external factors are a serious threat to the stability of the genome. Therefore, the cells have different molecular mechanisms for the efficient repair of this type of dá-mage. In this review we consider two main biochemical pathways of double-strand DNA breaks repair in eukaryotic cells--DNA strands nonhomologous end joining and homologous recombination between sister chromatids or chromatids of homologous chromosomes. Numerous data obtained recently for various eukaryotic cells suggest that complex interplay between the major DSB repair pathways normally facilitate the efficient repair and maintenance of the structural and functional genome integrity, but at the same time, under conditions ofgenotoxic factors exposure may induce increased genomic instability.

A novel bioorthogonal method for the modification of cells with single-stranded DNA oligomers is compared to five alternative methods with respect to labeling efficacy, specificity, and effects on cell viability. The new method is based on oxime ligation of aminooxybiotin to aldehyde groups installed by periodate cleavage of cell-surface glycans, followed by the coupling of preformed DNA-streptavidin conjugates. As compared with two literature-reported methods based on direct coupling of N-hydroxysuccinimidyl (NHS)-DNA or NHS-biotinylation as well as with techniques based on strain-promoted alkyne-azide cycloaddition, this method shows the highest labeling densities and is sufficiently mild to avoid cell damage. Functionality of the DNA tags is demonstrated by DNA-directed immobilization on solid substrates and assembly of small cell aggregates., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double-strand break signalling in human cells. Here, we compare the DNA-binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single-strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single-molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double-strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double-strand break resection and homologous recombination.

The neutral-neutral two-dimensional agarose gel technique is mainly used to determine the chromosomal positions where DNA replication starts, but it is also applied to visualize replication fork progression and breakage as well as intermediates in DNA recombination. Here we provide a step-by-step protocol to analyze the fairly underrepresented and fragile replication intermediates in yeast chromosomal DNA. The technique can also be adapted to analyze replication intermediates in chromosomal DNA of higher eukaryotic organisms.

The second parity rule of Chargaff (A≈T and G≈C within one strand) holds all over the living world with minor exceptions. It is maintained with higher accuracy for long sequences. The question addressed in the article is how different sequence types, with different biases from the parity, contribute to the general effect. It appears that the sequence segments with biases of opposite sign are intermingled, so that with sufficient sequence lengths the parity is established. The parity rule seems to be a cumulative result of a number of independent processes in the genome evolution, with the parity as their intrinsic property. Symmetrical appearance of simple repeats and of Alu sequences in the human DNA strands, and other contributions to the Chargaff parity II rule are discussed.

The ability to efficiently introduce DNA into cells is essential for many experiments in biology. This is an explanatory chapter providing an overview of the various methods for introducing DNA into bacteria, yeast, and mammalian cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The channel structure of the Ku protein elegantly reveals the mechanistic basis of sequence-independent DNA-end binding, which is essential to genome integrity after exposure to ionizing radiation or in V(D)J recombination. However, contradicting evidence indicates that this protein is also involved in the regulation of gene expression and in other regulatory processes with intact chromosomes. This computational study predicts that a putative DNA binding domain of this protein, the SAP domain, can form DNA-bound complexes with relatively high affinities (ΔG ≈ -20 kcal mol(-1)). The binding modes are searched by low frequency vibration modes driven by the fully flexible docking method while binding affinities are calculated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. We find this well defined 5 kDa domain with a helix-extended loop-helix structure is suitable to form favorable electrostatic and hydrophobic interactions with either the major groove or the minor groove of DNA. The calculation also reveals the sequence specified binding preference which may relate to the observed pause sites when Ku translocates along DNA and the perplex binding of Ku with circular DNA.

The general transcription factor IID (TFIID) is a key target for regulation because its binding to a core promoter is the nucleating step in transcription complex assembly. Many eukaryotic activators stimulate recruitment of the TFIID when its concentration is made limiting at a promoter in vitro. Magnesium-agarose gels can separate large complexes containing TFIID, TFIIA (the DA complex), and TFIIB (the DAB complex) and permit a quantitative measurement of how activators stimulate assembly of such complexes. The advantage of the electrophoretic mobility shift assay (EMSA) is that the reactions can be performed under subsaturating conditions where a TFIID footprint might not be observed. Typically, the activator is incubated with a 32P-labeled DNA template, recombinant TFIIA purified from Escherichia coli, and immunopurified TFIID. After incubation, the samples are electrophoresed on magnesium-containing agarose gels, dried onto DEAE-cellulose paper, and autoradiographed. The DNA-protein complexes containing TFIID migrate with reduced mobility on magnesium-agarose gels both because of the large size of the complex and because the TATA-binding protein (TBP) subunit induces a sharp bend in the DNA, causing altered mobility. By comparing the binding of TFIID over a wide concentration range, with and without activator, one can assess whether the activator interacts with TBP or with one of the TBP-associated factors (TAFIIs). Additional factors such as TFIIA and TFIIB can be added subsequently to quantify their contributions to assembly of the transcription complex.

In recent years geneticists have witnessed many significant observations which have seriously shaken the traditional concept of the gene. These specifically include the facts that (1) the boundaries of transcriptional units are far from clear; in fact, whole chromosomes if not the whole genome seem to be continuums of genetic transcription, (2) many examples of gene fusion are known, (3) likewise many examples of so-called encrypted genes are known in the organelle genomes of microbial eukaryotes and in prokaryotes, and (4) in addition to the structure of the gene, its functional status can also be inheritable, and, further, (5) epigenetic extra-genomic modes of inheritance, called genetic restoration, seem to be a rather common phenomenon, meaning that organisms can sometimes rewrite their DNA on the basis of RNA messages inherited from generations past. I will briefly review these observations and discuss the difficulties of defining the gene, and then formulate a new view, which is called the relational or systemic concept of the gene. It has to be noted that genes assume their information content characteristics in the Shannonian sense as nucleotide sequences of DNA (or RNA). However, on the basis of this we cannot say anything about their information content in the semantic sense. The semantic information content of genes is context-dependent. Genes namely assume their biochemical characteristics usually only within living cells, their developmental characteristics only within living organisms, and their evolutionary characteristics only within populations of living organisms.

Repeated elements can be widely abundant in eukaryotic genomes, composing more than 50% of the human genome, for example. It is possible to classify repeated sequences into two large families, "tandem repeats" and "dispersed repeats." Each of these two families can be itself divided into subfamilies. Dispersed repeats contain transposons, tRNA genes, and gene paralogues, whereas tandem repeats contain gene tandems, ribosomal DNA repeat arrays, and satellite DNA, itself subdivided into satellites, minisatellites, and microsatellites. Remarkably, the molecular mechanisms that create and propagate dispersed and tandem repeats are specific to each class and usually do not overlap. In the present review, we have chosen in the first section to describe the nature and distribution of dispersed and tandem repeats in eukaryotic genomes in the light of complete (or nearly complete) available genome sequences. In the second part, we focus on the molecular mechanisms responsible for the fast evolution of two specific classes of tandem repeats: minisatellites and microsatellites. Given that a growing number of human neurological disorders involve the expansion of a particular class of microsatellites, called trinucleotide repeats, a large part of the recent experimental work on microsatellites has focused on these particular repeats, and thus we also review the current knowledge in this area. Finally, we propose a unified definition for mini- and microsatellites that takes into account their biological properties and try to point out new directions that should be explored in a near future on our road to understanding the genetics of repeated sequences.

Background: The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed)., Results: We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens., Conclusion: In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.

Transposable elements are mobile genetic units that exhibit broad diversity in their structure and transposition mechanisms. Transposable elements occupy a large fraction of many eukaryotic genomes and their movement and accumulation represent a major force shaping the genes and genomes of almost all organisms. This review focuses on DNA-mediated or class 2 transposons and emphasizes how this class of elements is distinguished from other types of mobile elements in terms of their structure, amplification dynamics, and genomic effect. We provide an up-to-date outlook on the diversity and taxonomic distribution of all major types of DNA transposons in eukaryotes, including Helitrons and Mavericks. We discuss some of the evolutionary forces that influence their maintenance and diversification in various genomic environments. Finally, we highlight how the distinctive biological features of DNA transposons have contributed to shape genome architecture and led to the emergence of genetic innovations in different eukaryotic lineages.

Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments >or=1 kb with high specificity.

Contrary to mammalian DNA, which is thought to contain only 5-methylcytosine (m5C), bacterial DNA contains two additional methylated bases, namely N6-methyladenine (m6A), and N4-methylcytosine (m4C). However, if the main function of m5C and m4C in bacteria is protection against restriction enzymes, the roles of m6A are multiple and include, for example, the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA. Interestingly, even if adenine methylation is usually considered a bacterial DNA feature, the presence of m6A has been found in protist and plant DNAs. Furthermore, indirect evidence suggests the presence of m6A in mammal DNA, raising the possibility that this base has remained undetected due to the low sensitivity of the analytical methods used. This highlights the importance of considering m6A as the sixth element of DNA., (Copyright 2006 Wiley Periodicals, Inc.)

DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive description of these essential biochemical processes requires detailed understanding of helicase mechanisms. These enzymes are ubiquitous, having been identified in viruses, prokaryotes and eukaryotes. Disease states, such as xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome and Werner's syndrome, have been linked to defects in specific genes coding for DNA helicases. Helicases have been placed into different subfamilies based on sequence comparison. The largest subgroups are termed superfamily 1 and superfamily 2. A proposed mechanism for helicases in these classes has been described in terms of an 'inchworm model'. The inchworm model includes conformational changes driven by ATP binding and hydrolysis that allow unidirectional translocation along DNA. A monomeric form of the enzyme is proposed to have two DNA-binding sites that enable sequential steps of DNA binding and release. Significant differences exist between helicases in important aspects of the models such as the oligomerization state of the enzyme with some helicases functioning as monomers, some as dimers and others as higher-order oligomers.

Background: Nuclear genome size varies 300 000-fold, whereas transcriptome size varies merely 17-fold. In the largest genomes nearly all DNA is non-genic secondary DNA, mostly intergenic but also within introns. There is now compelling evidence that secondary DNA is functional, i.e. positively selected by organismal selection, not the purely neutral or 'selfish' outcome of mutation pressure. The skeletal DNA theory argued that nuclear volumes are genetically determined primarily by nuclear DNA amounts, modulated somewhat by genes affecting the degree of DNA packing or unfolding; the huge spread of nuclear genome sizes is the necessary consequence of the origin of the nuclear envelope and the nucleation of its assembly by DNA, plus the adaptively significant 300 000-fold range of cell volumes and selection for balanced growth by optimizing karyoplasmic volume ratios (essentially invariant with cell volume in growing/multiplying cells). This simple explanation of the C-value paradox is refined here in the light of new insights into the nature of heterochromatin and the nuclear lamina, the genetic control of cell volume, and large-scale eukaryote phylogeny, placing special emphasis on protist test cases of the basic principles of nuclear genome size evolution., Genome Miniaturization: and Expansion Intracellular parasites (e.g. Plasmodium, microsporidia) dwarfed their genomes by gene loss and eliminating virtually all secondary DNA. The primary driving forces for genome reduction are metabolic and spatial economy and cell multiplication speed. Most extreme nuclear shrinkage yielded genomes as tiny as 0.38 Mb (making the nuclear genome size range effectively 1.8 million-fold!) in some minute enslaved nuclei (nucleomorphs) of cryptomonads and chlorarachneans, chimaeric cells that also retain a separate normal large nucleus. The latter shows typical correlation between genome size and cell volume, but nucleomorphs do not despite co-existing in the same cell for >500 My. Thus mutation pressure does not inexorably increase genome size; selection can eliminate essentially all non-coding DNA if need be. Nucleomorphs and microsporidia even reduced gene size. Expansion of secondary DNA in the main nucleus, and in large-celled eukaryotes generally, must be positively selected for function. Ciliate nuclear dimorphism provides a key test that refutes the selfish DNA and strongly supports the skeletal DNA/karyoplasmic ratio interpretation of genome size evolution., Genetic Control of Cell Volume Is Multigenic: The quantitatively proportional correlation between genome size and cell size cannot be explained by purely mutational theories, as eukaryote cell volumes are causally determined by cell cycle control genes, not by DNA amounts.

We continue our study, Poland [Biophysical Chemistry 110 (2004) 59-2], of the distribution of C or G (C-G for short) in the DNA of select organisms, in particular, the tendency for C-G to cluster on all scales with respect to the number of bases considered. We previously found that if we counted the number of C-G bases in consecutive, nonoverlapping boxes containing a total of m bases, then the width of the distribution function describing how many C-G bases are in a box increases with respect to m dramatically relative to the width expected for a random distribution. The relative width of the C-G composition distribution function was found to vary accurately as a power law with respect to m, the size of the box, over a very wide range of m values. We express the power law in terms of a characteristic exponent gamma, that is, the relative widths of the distributions vary as m(gamma). The enhanced relative width of the distribution functions is a direct consequence of the tendency for boxes of similar composition to follow one another. This tendency represents persistence in composition from box to box and hence we refer to gamma as the persistence exponent. The occurrence of a power law means that the tendency for C-G to cluster is present on all scales of sequence length (box size) up to the total length of the chromosome which for bacteria is the entire genome. The persistence exponent gamma that characterizes the power law is thus an important parameter describing the distribution of C-G on all scales from individual base pairs up to the total length of the DNA sample considered. In the present paper, we determine the characteristic exponent gamma and the associated fractal dimension of DNA samples for a selection of species representing all of the major types of organism, that is, we explore the phylogeny of the exponent gamma. Here we treat six prokaryotes and six eukaryotes which, together with the species we have previously treated, brings the total number of species we have examined to 15. We find the power law form for the C-G distribution for all of the species treated and hence this behavior seems to be ubiquitous. The values of the characteristic exponent gamma that we find tend to cluster around the value gamma=0.20 with no obvious pattern with respect to phylogeny. The extreme values that we obtain are gamma=0.057 (yeast) and gamma=0.386 (human). We conclude by showing that the persistence of C-G clustering on the scale of the length of a chromosome is dramatically illustrated by interpreting the C-G distribution as a random walk.

The melting of the coding and non-coding classes of natural DNA sequences was investigated using a program, MELTSIM, which simulates DNA melting based upon an empirically parameterized nearest neighbor thermodynamic model. We calculated T(m) results of 8144 natural sequences from 28 eukaryotic organisms of varying F(GC) (mole fraction of G and C) and of 3775 coding and 3297 non-coding sequences derived from those natural sequences. These data demonstrated that the T(m) vs. F(GC) relationships in coding and non-coding DNAs are both linear but have a statistically significant difference (6.6%) in their slopes. These relationships are significantly different from the T(m) vs. F(GC) relationship embodied in the classical Marmur-Schildkraut-Doty (MSD) equation for the intact long natural sequences. By analyzing the simulation results from various base shufflings of the original DNAs and the average nearest neighbor frequencies of those natural sequences across the F(GC) range, we showed that these differences in the T(m) vs. F(GC) relationships are largely a direct result of systematic F(GC)-dependent biases in nearest neighbor frequencies for those two different DNA classes. Those differences in the T(m) vs. F(GC) relationships and biases in nearest neighbor frequencies also appear between the sequences from multicellular and unicellular organisms in the same coding or non-coding classes, albeit of smaller but significant magnitudes.

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

Both DNA replication and transcription are structurally asymmetric processes. An asymmetric nucleotide substitution pattern has been observed between the leading and the lagging strand, and between the coding and the noncoding strand, in eubacterial, viral, and organelle genomes. Similar studies in eukaryotes have been rare, because the origins of replication in nuclear genomes are mostly unknown and the replicons are much shorter than those of prokaryotes. To circumvent these predicaments, all possible pairs of neighboring genes that are located on different strands of nuclear DNA were selected from the complete genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum, Encephalitozoon cuniculi, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Mus musculus, and Homo sapiens. For such a pair of genes, one is likely coded from the leading strand and the other from the lagging strand. By examining the introns and the fourfold degenerate sites of codons in the genes of each pair, we found that the relative frequencies of T vs. A and of G vs. C are significantly skewed in most eukaryotes studied. In a gene pair, the potential effects of replication- and transcription-associated mutation bias on strand asymmetry are in the same direction for one gene where leading strand synthesis shares the same template with transcription, while they tend to be canceled out in the other gene. Our study demonstrates that DNA replication-associated and transcription-associated mutation bias and/or selective codon usage bias may affect the strand nucleotide composition asymmetrically in eukaryotic genomes.

We introduce a novel, linguistic-like method of genome analysis. We propose a natural approach to characterizing genomic sequences based on occurrences of fixed length words from a predefined, sufficiently large set of words (strings over the alphabet [A, C, G, T]). A measure based on this approach is called compositional spectrum and is actually a histogram of imperfect word occurrences. Our results assert that the compositional spectrum is an overall characteristic of a long sequence i.e., a complete genome or an uninterrupted part of a chromosome. This attribute is manifested in the similarity of spectra obtained on different stretches of the same genome, and simultaneously in a broad range of dissimilarities between spectral representations of different genomes. High flexibility characterizes this approach due to imperfect matching and as a result sets of relatively long words can be considered. The proposed approach may have various applications in intra- and intergenomic sequence comparisons.

The in vitro effect of tetrahydrocortisol-apolipoprotein A-I complex on native adult rat liver DNA results in the formation of S1 nuclease sensitive fragments that are irregularly distributed throughout a genome. Low-angle X-ray scattering showed that after the interaction with the tetrahydrocortisol-apolipoprotein A-I complex, DNA can bind to RNA-polymerase with a high and dose-dependent cooperativity. This indicates that the effect of tetrahydrocortisol-apolipoprotein A-I complex on secondary eukaryotic DNA structure causes a local denaturation of the double helix, promoting high cooperativity of binding to RNA-polymerase. The reduced form of the hormone, tetrahydrocortisol, previously considered as an inactive metabolite, when complexed with apolipoprotein A-I, promotes a biological function similar to that of a transcription factor.

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.

The sequencing of several genomes from each of the three domains of life (Archaea, Bacteria and Eukarya) has provided a huge amount of data that can be used to gain insight about early cellular evolution. Some features of the universal tree of life based on rRNA polygenies have been confirmed, such as the division of the cellular living world into three domains. The monophyly of each domain is supported by comparative genomics. However, the hyperthermophilic nature of the 'last universal common ancestor' (LUCA) is not confirmed. Comparative genomics has revealed that gene transfers have been (and still are) very frequent in genome evolution. Nevertheless, a core of informational genes appears more resistant to transfer, testifying for a close relationship between archaeal and eukaryal informational processes. This observation can be explained either by a common unique history between Archaea and Eukarya or by an atypical evolution of these systems in Bacteria. At the moment, comparative genomics still does not allow to choose between a simple LUCA, possibly with an RNA genome, or a complex LUCA, with a DNA genome and informational mechanisms similar to those of Archaea and Eukarya. Further comparative studies on informational mechanisms in the three domains should help to resolve this critical question. The role of viruses in the origin and evolution of DNA genomes also appears an area worth of active investigations. I suggest here that DNA and DNA replication mechanisms appeared first in the virus world before being transferred into cellular organisms.

Motivation: A rapid growth in the number of genes with known sequences calls for developing automated tools for their classification and analysis. It became clear that nucleosome packaging of eukaryotic DNA is very important for gene functioning. Automated computer tools for characterization of nucleosome packaging density could be useful for studying of gene regulation and genome annotation., Results: A program for constructing nucleosome formation potential profiles of eukaryotic DNA sequences was developed. Nucleosome packaging density was analyzed for different functional types of human promoters. It was found that in promoters of tissue-specific genes, the nucleosome formation potential was essentially higher than in genes expressed in many tissues, or housekeeping genes. Hence, capability of nucleosome positioning in the promoter region may serve as a factor regulating gene expression., Availability: The program for nucleosome sites recognition is included into the GeneExpress system; section 'DNA Nucleosomal Organization', http://wwwmgs.bionet.nsc.ru/mgs/programs/recon/.

Motivation: Sequencing of complete eukaryotic genomes and large syntenic fragments of genomes makes it possible to apply genomic comparison for gene recognition., Results: This paper describes a spliced alignment algorithm that aligns candidate exon chains of two homologous genomic sequence fragments from different species. The algorithm is implemented in Pro-Gen software. Unlike other algorithms, Pro-Gen does not assume conservation of the exon-intron structure. Amino acid sequences obtained by the formal translation of candidate exons are aligned instead of nucleotide sequences, which allows for distant comparisons. The algorithm was tested on a sample of human-mammal (mouse), human-vertebrate (Xenopus ) and human-invertebrate (Drosophila ) gene pairs. Surprisingly, the best results, 97-98% correlation between the actual and predicted genes, were obtained for more distant comparisons, whereas the correlation on the human-mouse sample was only 93%. The latter value increases to 95% if conservation of the exon-intron structure is assumed. This is caused by a large amount of sequence conservation in non-coding regions of the human and mouse genes probably due to regulatory elements., Availability: Pro-Gen v. 3.0 is available to academic researchers free of charge at http://www.anchorgen.com/pro_gen/pro_gen.html.

We use the "wavelet transform microscope" to carry out a comparative statistical analysis of DNA bending profiles and of the corresponding DNA texts. In the three kingdoms, one reveals on both signals a characteristic scale of 100-200 bp that separates two different regimes of power-law correlations (PLC). In the small-scale regime, PLC are observed in eukaryotic, in double-strand DNA viral, and in archaeal genomes, which contrasts with their total absence in the genomes of eubacteria and their viruses. This strongly suggests that small-scale PLC are related to the mechanisms underlying the wrapping of DNA in the nucleosomal structure. We further speculate that the large scale PLC are the signature of the higher-order structure and dynamics of chromatin.

Eukaryotic promoters often contain a bent DNA structure, suggesting that this structure plays some role in transcription. To reveal the role, we need more information on the promoters that contain or flank a bent DNA structure. In this study, we collected such promoters by the following approach: we first isolated human genomic DNA fragments that contained at least one bent DNA structure, then shotgun cloned them into a promoter trap vector, screened DNA fragments that functioned as a promoter, and finally found the promoters of interest by determining the bent DNA locus and the region expressing promoter activity. From 1,187 recombinant plasmids, we isolated 51 that showed promoter activity. Structural and functional analyses of randomly selected 10 clones with inserts of 548-913 bp demonstrated 11 sequences that could drive transcription. Unexpectedly, all of these clones met our purpose: i.e., each segment that showed a promoter activity (67-179 bp) was very close to the bent DNA structure (spanning about 150 bp in all clones), and in some cases overlapped it. More interestingly, these bent DNA structures all had a superhelical writhe. We propose a hypothesis that in the bent-DNA-containing eukaryotic promoters. bent DNA organizes local chromatin infrastructure appropriately for transcription initiation.

Based on worm like chain model, DNA structural parameters--tilt, roll and rise, derived from crystallographic database have been used to determine the flexibility of DNA that regulates the nucleosomal translational positioning. Theoretically derived data has been compared to the experimental values available in loshikhes and Trifonov's database. The methodology has been extended to determine the flexibility of 18S rRNA genome in eukarya, where yeast shows a distinct difference when compared with mammals like human, mouse and rabbit.

We evaluated the utility of three in vitro methods to monitor the biological potency of PLGA encapsulated DNA. For each assay we also determined whether the biological activity was influenced by the structural profile of DNA isomers. Collectively, the results indicate that all three methods can be used to evaluate the biological activity of DNA extracted from PLGA microspheres, but they are differentially sensitive to the structural changes of plasmid DNA that can occur during microencapsulation and microsphere storage. More specifically, mammalian cell transfection followed by an enzyme assay affords an accurate determination of DNA potency over time and is less influenced by DNA isoform than bacterial transformation. Cell-free transcription/translation systems can also be utilized, and the results of this assay are influenced by DNA isoform. Finally, bacterial transformation was found to be more sensitive to DNA isoform than the other assay methods.

Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages., (Copyright 2000 Academic Press.)

Many members of the SWI2/SNF2 family of adenosine triphosphatases participate in the assembly/disassembly of multiprotein complexes involved in the DNA metabolic processes of transcription, recombination, repair, and chromatin remodeling. The DNA molecule serves as an essential effector or catalyst for most of the members of this particular class of proteins, and the structure of the DNA may be more important than the nucleotide sequence. Inspection of the DNA structure at sites where multiprotein complexes are assembled/disassembled for these various DNA metabolic processes reveals the presence of a common element: a double-stranded to single-stranded transition region. We now show that this DNA element is crucial for the ATP hydrolytic function of an SWI2/SNF2 family member: DNA-dependent ATPase A. We further demonstrate that a domain containing the seven helicase-related motifs that are common to the SWI2/SNF2 family of proteins mediates the interaction with the DNA, yielding specific DNA structural recognition. This study forms a primary step toward understanding the physico-biochemical nature of the interaction between a particular class of DNA-dependent ATPase and their DNA effectors. Furthermore, this study provides a foundation for development of mechanisms to specifically target this class of DNA-dependent ATPases.

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.

Genic DNA functions are commonplace: coding for proteins and specifying non-messenger RNA structure. Yet most DNA in the biosphere is non-genic, existing in nuclei as non-coding or secondary DNA. Why so much secondary DNA exists and why its amount per genome varies over orders of magnitude (correlating positively with cell volume) are central biological problems. A novel perspective on secondary DNA function comes from natural eukaryote eukaryote chimaeras (cryptomonads and chlorarachneans) where two phylogenetically distinct nuclei have coevolved within one cell for hundreds of millions of years. By comparing cryptomonad species differing 13-fold in cell volume, we show that nuclear and nucleomorph genome sizes obey fundamentally different scaling laws. Following a more than 125-fold reduction in DNA content, nucleomorph genomes exhibit little variation in size. Furthermore, the present lack of significant amounts of nucleomorph secondary DNA confirms that selection can readily eliminate functionless nuclear DNA, refuting 'selfish' and 'junk' theories of secondary DNA. Cryptomonad nuclear DNA content varied 12-fold: as in other eukaryotes, larger cells have extra DNA, which is almost certainly secondary DNA positively selected for a volume-related function. The skeletal DNA theory explains why nuclear genome size increases with cell volume and, using new evidence on nucleomorph gene functions, why nucleomorph genomes do not.

Motivation: We describe a new content-based approach for the detection of promoter regions of eukaryotic protein encoding genes. Our system is based on three interpolated Markov chains (IMCs) of different order which are trained on coding, non-coding and promoter sequences. It was recently shown that the interpolation of Markov chains leads to stable parameters and improves on the results in microbial gene finding (Salzberg et al., Nucleic Acids Res., 26, 544-548, 1998). Here, we present new methods for an automated estimation of optimal interpolation parameters and show how the IMCs can be applied to detect promoters in contiguous DNA sequences. Our interpolation approach can also be employed to obtain a reliable scoring function for human coding DNA regions, and the trained models can easily be incorporated in the general framework for gene recognition systems., Results: A 5-fold cross-validation evaluation of our IMC approach on a representative sequence set yielded a mean correlation coefficient of 0.84 (promoter versus coding sequences) and 0.53 (promoter versus non-coding sequences). Applied to the task of eukaryotic promoter region identification in genomic DNA sequences, our classifier identifies 50% of the promoter regions in the sequences used in the most recent review and comparison by Fickett and Hatzigeorgiou ( Genome Res., 7, 861-878, 1997), while having a false-positive rate of 1/849 bp.

In eukaryotic cell nuclei the genome is organized into large loops attached to the nuclear matrix. Rearrangements of the genome frequently occur via an illegitimate recombination between loop anchorage sites resulting in deletion or repositioning of DNA loops. The illegitimate recombination between loop anchorage sites is possibly mediated by topoisomerase II. Treatments stabilizing intermediate covalent complexes of topoisomerase II with DNA seem to increase the possibility of illegitimate recombination between loop anchorage regions. On the basis of these and some other observations we suggest that chromosomal DNA loops constitute basic units of the genome evolution, or, in other words, structural blocks of the eukaryotic genome.

DNA can be divided functionally into three categories: (1) genes--which code for proteins or specify non-messenger RNAs; (2) semons--short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA--which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether 'junk' or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago. In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively. The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection. The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.