Article

# The Twisted Circular Form of Polyoma Viral DNA

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## Abstract

The major part of the DNA from polyoma virus has been shown to consist of circular base-paired duplex molecules without chain ends.(1-3) The intertwined circular form accounts for the ease of renaturation(4) of this DNA and the failure of the strands to separate in strand-separating solvents.(1-3)

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... Torsional stress must be disentangled along with the transient breaking and rejoining of DNA strands (Champoux 2001;Wang 2002;Corbett and Berger 2004) to ensure the normal essential cellular processes, such as transcription, replication, recombination and other essential DNA processes (Topcu 2001;Nitiss 1998;Wu et al. 1988), while the solving of topological questions has brought tremendous challenges. Until the discoveries of the DNA supercoiling by Jerome Vinograd in 1965(Vinograd et al. 1965) and the first DNA Topo (x protein, namely Topo I/type I) by Wang (1971) from Escherichia coli (E.coli), these challenges mentioned above were gradually overcome for a long time with tremendous difficulties. Subsequently, numerous studies focusing on DNA Topos have led to the discovery of multiple classes of DNA Topos (Table 1). ...
... Torsional stress must be disentangled along with the transient breaking and rejoining of DNA strands (Champoux 2001;Wang 2002;Corbett and Berger 2004) to ensure the normal essential cellular processes, such as transcription, replication, recombination and other essential DNA processes (Topcu 2001;Nitiss 1998;Wu et al. 1988), while the solving of topological questions has brought tremendous challenges. Until the discoveries of the DNA supercoiling by Jerome Vinograd in 1965(Vinograd et al. 1965) and the first DNA Topo (x protein, namely Topo I/type I) by Wang (1971) from Escherichia coli (E.coli), these challenges mentioned above were gradually overcome for a long time with tremendous difficulties. Subsequently, numerous studies focusing on DNA Topos have led to the discovery of multiple classes of DNA Topos (Table 1). ...
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The continuing rise in cancer morbidity and mortality all over the world makes cancer the No. 2 cause of death in humans, posing a major public health challenge. Currently, surgery, radiotherapy, and chemotherapy are the three major approaches to treat cancer. However, the disadvantages of serious side effects, and poor therapeutic effect of the aforementioned methods in many clinical cases have become a burning question to be solved. DNA topoisomerases (Topos) are the key enzymes for DNA replication, transcription, recombination and repair, and have been regarded as target enzymes for some anticancer drugs, leading to the development of some successful anticancer drugs now in clinical use. Moreover, medicinal plants with numerous bioactive compounds are one of the most important sources for the discovery and development of anticancer drugs. In an active effort to search for Topo inhibitors from medicinal plants, affinity selection–ultrafiltration/high performance liquid chromatography–mass spectrometry (AS/UF–LC–MS) is among the most efficient techniques in term of sensitivity, throughput, and wide applicability. This review summarized the most recent advances in screening Topo inhibitors with AS/UF–LC–MS from medicinal plants over the past 15 years, with focus on structures, functions, the mechanisms of action of Topos and the procedures of the AS/UF–LC–MS technology, with an aim to provide comprehensive insights for new drug research and the discovery of more effective leading compounds against Topos.
... Circular DNA isolated from polyoma virus shows many crossings of the DNA double strands, indicating that the DNA is supercoiled. Reproduced from Vinograd et al. (1965) cell, DNA is associated with a vast array of proteins -for example, proteins required for its replication, its transcription into RNA and its packaging within the cell. Many of these proteins wrap DNA around themselves, or in other ways constrain or bend the DNA molecule. ...
... In 1965 Jerome Vinograd and his colleagues suggested that closed-circular DNA molecules could adopt a 'twisted circular form' (Vinograd et al., 1965). Such a form would result if, before joining the ends of a linear duplex DNA into a closed circle, one end was twisted relative to the other to introduce some strain into the molecule. ...
... Ring polymers in solution have attracted much interest in various branches of science [1,2,3]. Ring polymers with trivial topology are observed in nature such as circular DNA [4], while circular DNA with nontrivial knot types are derived in experiments [5,6]. Topological structures related to knots or pseudo-knots have been discussed in association with protein folding [7,8]. ...
... For the figureeight knot (4 1 ) the ratio of topological swelling becomes 1.0 at N = 291, which is slightly larger than N 0 . 4 Scaling behavior of SAP with a fixed knot 4 ...
Article
We show that the average size of self-avoiding polygons (SAP) with a fixed knot is much larger than that of no topological constraint if the excluded volume is small and the number of segments is large. We call it topological swelling. We argue an "enhancement" of the scaling exponent for random polygons with a fixed knot. We study them systematically through SAP consisting of hard cylindrical segments with various different values of the radius of segments. Here we mean by the average size the mean-square radius of gyration. Furthermore, we show numerically that the equilibrium length of a composite knot is given by the sum of those of all constituent prime knots. Here we define the equilibrium length of a knot by such a number of segments that topological entropic repulsions are balanced with the knot complexity in the average size. The additivity suggests the local knot picture.
... The various configurations taken up by DNA molecules, which has formed the basis for much of the structural studies of bacterial plasmids, were previously well investigated and characterized by Sinsheimer and coworkers (27,79) from studies with the doublestranded replicative form of the singlestranded phage, OX174, and by Vinograd and co-workers (229) in their experiments with polyoma virus. They have shown (Fig. 4) (193). ...
... The identity of the CCC and OC peaks can be confirmed by taking advantage of the fact that a single scission or nick in just one of the strands of a CCC molecule produces an OC molecule (Fig. 4). This may be done by one of several methods, including limiting deoxyribonuclease treatment (76,79,128,229) or Xirradiation (85), and leads to a decrease in the size of the CCC peak and a corresponding increase in the size of the OC peak which are thereby defined. The absolute value of the sedimentation coefficient for the OC peak may be determined by cosedimentation of a carefully purified DNA standard of known sedimentation coefficient and by application of the formula, S1IS2 = D1/D2 (26), where S1, S2 are the sedimentation coefficients and D1, D2 the distances of the peaks from the meniscus of the OC and standard peaks, respectively. ...
... Early studies of DNA plasmids using ultracentrifugation approaches showed that different structures were present in a sample containing only plasmids of equal molecular weight [24][25][26][27]. The nature of this structural difference was determined by Vinograd et al. [28], who showed that a single-strand nick triggered the sedimentation of a single species, indicating that DNA normally had a constrained 'twisted circular structure'. Using sucrose-gradient sedimentation, together with the DNA intercalator ethidium bromide, it was possible to accurately determine the number of supercoils within a plasmid DNA sample through a laborious titration approach [29]. ...
Article
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Supercoiling is a fundamental property of DNA, generated by polymerases and other DNA-binding proteins as a consequence of separating/bending the DNA double helix. DNA supercoiling plays a key role in gene expression and genome organization, but has proved difficult to study in eukaryotes because of the large, complex and chromatinized genomes. Key approaches to study DNA supercoiling in eukaryotes are (1) centrifugation-based or electrophoresis-based techniques in which supercoiled plasmids extracted from eukaryotic cells form a compacted writhed structure that migrates at a rate proportional to the level of DNA supercoiling; (2) in vivo approaches based on the preferential intercalation of psoralen molecules into under-wound DNA. Here, we outline the principles behind these techniques and discuss key discoveries, which have confirmed the presence and functional potential of unconstrained DNA supercoiling in eukaryotic genomes.
... Statistical and dynamical properties of polymers with nontrivial structures in chemical connectivity and those derived from topological constraints such as ring polymers with fixed knots have attracted much interest in several branches of physics, chemistry and biology. Although some properties of ring polymers in solution were studied rather early, such as in the 1940s and 1960s [9][10][11][12], circular DNA had been found in nature in the 1960s, and knotted DNA molecules are synthesized in experiments in the 1980s [2,[13][14][15]; looped or knotted proteins had been found in nature during the 2000s [5,16]. Here, we recall that the three-dimensional conformation of a ring polymer in solution is described by a closed curve with no ends, and its topology is represented by a knot. ...
Article
We review recent theoretical studies on the statistical and dynamical properties of polymers with nontrivial structures in chemical connectivity and those of polymers with a nontrivial topology, such as knotted ring polymers in solution. We call polymers with nontrivial structures in chemical connectivity expressed by graphs "topological polymers". Graphs with no loop have only trivial topology, while graphs with loops such as multiple-rings may have nontrivial topology of spatial graphs as embeddings in three dimensions, e.g., knots or links in some loops. We thus call also such polymers with nontrivial topology "topological polymers", for simplicity. For various polymers with different structures in chemical connectivity, we numerically evaluate the mean-square radius of gyration and the hydrodynamic radius systematically through simulation. We evaluate the ratio of the gyration radius to the hydrodynamic radius, which we expect to be universal from the viewpoint of the renormalization group. Furthermore, we show that the short-distance intrachain correlation is much enhanced for real topological polymers (the Kremer-Grest model) expressed with complex graphs. We then address topological properties of ring polymers in solution. We define the knotting probability of a knot K by the probability that a given random polygon or self-avoiding polygon of N vertices has the knot K. We show a formula for expressing it as a function of the number of segments N, which gives good fitted curves to the data of the knotting probability versus N. We show numerically that the average size of self-avoiding polygons with a fixed knot can be much larger than that of no topological constraint if the excluded volume is small. We call it "topological swelling".
... Despite the abundant information on their replication modes and requirements [1][2][3][4][5] little is known about how the topology of the DNA changes as the molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. This is particularly intriguing since SV40 and EBV were among the first viral DNAs where DNA supercoiling and catenation were originally discovered [12][13][14][15]. Moreover, Keller [16] was the first to use agarose gel electrophoresis combined with intercalating agents to determine the exact number of superhelical turns in SV40 DNA. ...
Article
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Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3’Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV “family of repeats” induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner.
... Control of DNA supercoiling is of vital importance to cells. Torsional strain imposed by DNA- 31 processing enzymes induces supercoiling of DNA, which triggers large structural rearrangements 32 through the formation of plectonemes (Vinograd et al., 1965). Recent biochemical studies 33 suggest that supercoiling plays an important role in the regulation of gene expression in both 34 prokaryotes (Le et al., 2013) and eukaryotes (Naughton et al., 2013;Pasi and Lavery, 2016). ...
Article
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The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentaly confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.
... Today, we understand that DNA is topologically polymorphic, that is, it can exist in many different structural forms. One of these forms, the supercoil, was first illustrated by Vinograd and co-workers [61] in EM micrographs of circular DNA from polyoma virus. These micrographs revealed the presence of multiple intertwined loops. ...
Article
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The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.
... Variable DNA supercoiling is a fundamental principle in the control of gene expression in bacteria [1][2][3][4]. DNA is usually negatively supercoiled in bacterial cells because it contains a deficit of helical turns [5][6][7]. In its B form, the strands of the DNA duplex make one complete turn every 10.5 base pairs. ...
Article
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Background: The processes of DNA supercoiling and transcription are interdependent because the movement of a transcription elongation complex simultaneously induces under- and overwinding of the DNA duplex and because the initiation, elongation and termination steps of transcription are all sensitive to the topological state of the DNA. Results: Policing of the local and global supercoiling of DNA by topoisomerases helps to sustain the major DNA-based transactions by eliminating barriers to the movement of transcription complexes and replisomes. Recent data from whole-genome and single-molecule studies have provided new insights into how interactions between transcription and the supercoiling of DNA influence the architecture of the chromosome and how they create cell-to-cell diversity at the level of gene expression through transcription bursting. Conclusions: These insights into fundamental molecular processes reveal mechanisms by which bacteria can prevail in unpredictable and often hostile environments by becoming unpredictable themselves.
... The authors declare that they have no competing interests Conter, A. et al. (1997) Box 1. DNA in bacterial cells is maintained in an underwound state due to a deficit in linking number, the number of times that one DNA strand in the duplex crosses the other, compared with fully relaxed DNA (Bauer et al., 1980;Boles et al., 1990;Vinograd et al., 1965). This deficiency introduced torsional stress into DNA and the molecule seeks to adopt a minimum energy conformation through the writhing of the axis of the already-coiled DNA duplex ('supercoiling'), by loss of DNA twist through local unzipping of the two DNA strands, or some combination of the two (Sinden, 1994). ...
Article
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In this opinion article we highlight links between the H-NS nucleoid-associated protein, variable DNA topology, the regulation of CRISPR-cas locus expression, CRISPR-Cas activity, and the recruitment of novel genetic information by the CRISPR array. We propose that the requirement that the invading mobile genetic element be negatively supercoiled limits effective CRISPR action to a window in the bacterial growth cycle when DNA topology is optimal, and that this same window is used for the efficient integration of new spacer sequences at the CRISPR array. H-NS silences CRISPR promoters, and we propose that antagonists of H-NS, such as the LeuO transcription factor, provide a basis for a stochastic genetic switch that acts at random in each cell in the bacterial population. In addition, we wish to propose a mechanism by which mobile genetic elements can suppress CRISPR-cas transcription using H-NS homologues. Although the individual components of this network are known, we propose a new model in which they are integrated and linked to the physiological state of the bacterium. The model provides a basis for cell-to-cell variation in the expression and performance of CRISPR systems in bacterial populations.
... DNA superhelicity, discovered over fifty years ago (1)(2)(3), is an essential physical property of the DNA double helix that can be most easily understood for closed circular duplex DNA (ccDNA) molecules such as plasmids. Each strand in a ccDNA is a circle, and these two circles are interlinked due to the helical nature of DNA Superhelicity can also be imposed on noncircular DNA molecules. ...
Article
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R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops’ potential biological roles under both normal and aberrant conditions.
... In contrast, DNA stores a lot of binary information due to the presence of multiple helices. Hence, for deployment from a traditional DNA transmission model into a stochastic DNA transmission model, the leading barriers are the channel construction 10 , the hybrid nature of helix direction 11,12 , the limitation of information bound 13,14 and the traditional source distribution method 15,16 . To effectively deal with these impairments, a black hole-aided communication model can offer a benefit for spontaneously designing a stochastic DNA transmission model and achieving integrated information bound. ...
Preprint
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This paper presents a black hole-aided deep-helix stochastic symmetric (termed as BH-DHSS) channel to enrich the traditional source distribution method and achieve integrated information bound to Deoxyribonucleic acid (DNA) communications. The conventional Bernoulli distributions and Shannon bounds do not match perfectly with the DNA information bounds because of multiple-helix directional issues. Consequently, we propose a BH-DHSS channel model in this paper. The proposed BH-DHSS channel model follows a similar fashion to the Watson-Crick DNA model and incorporates all the earlier DNA models into a stochastic DNA model for measuring an integrated entropy and capacity bound. To successfully design the proposed BH-DHSS channel, we first define a black hole-aided Bernoulli distribution method and then consider the BH-DHSS channel model. Following the geometric and graphical insights of the proposed BH-DHSS channel, we then demonstrate the resemblance between the DNA physical structure and the galaxy layout. As we explore this channel, we find that BH-DHSS shapes a deep-pair-ellipse geometrically when a deep-pair bit or digit is distributed. In addition, the proposed channel graphically shapes as a beautiful circulant ring. This ring has a hole in the center that looks like a central black hole in a galaxy. The coordinates of the inner ellipses denote a deep-double helix, and the coordinates of the outer-ellipses sketch a deep-parallel strand. Finally, the proposed BH-DHSS channel significantly outperforms the traditional binary symmetric channel and is verified by computer simulations in terms of Shannon entropy and capacity bound.
... This is similar to the Shope rabbit papilloma virus DNA, but differs from polyoma virus DNA (35). The estimated base composition of the virus DNA, on the basis of buoyant density in a cesium chloride equilibrium density gradient relative to marker DNA (139), was found to be 41% guanine plus cytosine. of the molecule, and that the 28S component has Under the same conditions, canine, bovine, and a supercoiled configuration (Fig. 1), such as has rabbit papilloma virus DNA preparations were recently been proposed for the fast component of found to contain 43, 45.5, and 47% guanine plus polyoma, virus (147). On the basis of the sedicytosine, respectively, and polyoma DNA, 48% mentation coefficient of the 18S component, the (35). ...
... Twenty years ago hydrodynamic studies by Vinograd and co-workers (160) showed that closed, circular DNA molecules are more compact than their nicked or linear counterparts. When examined by electron microscopy, the closed, circular DNA molecules appeared to be twisted (166). ...
... It is to be noted that a black or a white hole is a constant blind source dependence natural strategy, which is widely used to expand the space of a region and help execute a multiple-helix directional issue in the part of nature. Since DNA 1,2,3,4 is a part of nature, it carries out a multiple-helix 8,9 directional issue. Hence, a black-hole strategy is not only present in the Galaxy, its existence is also noticeable in the DNA or other parts of nature such as a Cyclone. ...
Preprint
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In this article, we present a black-hole -aided deep -helix (bh-dh) channel model to enhance information bound and mitigate a multiple-helix directional issue in Deoxyribonucleic acid (DNA) communications. The recent observations of DNA do not match with Shannon bound due to their multiple-helix directional issue. Hence, we propose a bh-dh channel model in this paper. The proposed bh-dh channel model follows a similar fashion of DNA and enriches the earlier DNA observations as well as achieving a composite like information bound. To do successfully the proposed bh-dh channel model, we first define a black-hole -aided Bernoulli -process and then consider a symmetric bh-dh channel model. After that, the geometric and graphical insight shows the resemblance of the proposed bh-dh channel model in DNA and Galaxy layout. In our exploration, the proposed bh-dh symmetric channel geometrically sketches a deep-pair-ellipse when a deep-pair information bit or digit is distributed in the proposed channel. Furthermore, the proposed channel graphically shapes as a beautiful circulant ring . The ring contains a central-hole , which looks like a central-black-hole of a Galaxy. The coordinates of the inner-ellipses denote a deep-double helix, and the coordinates of the outer-ellipses sketch a deep-parallel strand. Finally, the proposed bh-dh symmetric channel significantly outperforms the traditional binary-symmetric channel and is verified by computer simulations in terms of Shannon entropy and capacity bound.
... This technique was first described by Vinograd et al. (1965) and it was improved as the alkalinehalo assay by Sestili et al. (2006). In the normal comet assay, and in response to the electric current, charged DNA migrates away from the nucleus. ...
Thesis
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Chapter
The helical structure of DNA was proposed in 1953 by Watson and Crick (1). Twelve years later, Vinograd and collaborators (2–4) found that the helix axis can also be coiled in circular DNA. This structure was called supercoiling. This finding was extended to linear DNA by Pettijohn and others (5–8). It is now known that practically all DNA in vivo is supercoiled (9). Because most of the functions of DNA require untwisting, the importance of the enzymes required for this is self-evident. The first such enzyme identified was called the protein ω (10).It is now called topoisomerase I (top1) because in untwisting the supercoiled DNA, it breaks only one of the two strands, whereas topoisomerase II (top2), initially called gyrase, breaks both. Topoisomerase type I enzymes are ubiquitous, having been found in every prokaryotic or eukaryotic cells investigated so far with the exception of sea urchin sperm (11).
Chapter
This chapter deals with the classical hydrodynamic theory of steady-state transport properties, such as the translational friction and diffusion coefficients and intrinsic viscosity, of the unperturbed HW chain, including the KP wormlike chain as a special case, on the basis of the cylinder and touched-bead models. An analysis of experimental data is made from various points of view, which are based on the present theory, especially for flexible polymers. In the same spirit as that in Chap. 5, use is then made of experimental data obtained for several flexible polymers in the $$\varTheta$$ state over a wide range of molecular weight, including the oligomer region, and also for typical semiflexible polymers. As a result, it is pointed out that there still remain several unsolved problems for flexible polymers even in the unperturbed state. It is convenient to begin by giving a general consideration of some aspects of polymer hydrodynamics which leads to the adoption of the present hydrodynamic models.
Chapter
The principal members of the papova group are polyoma virus (Stewart et al., 1957), simian virus 40 (SV40), which is a vacuolating virus of monkeys (Sweet and Hilleman, 1960), and the papilloma viruses (Melnick, 1962). The name for this group of viruses is derived from the first two letters of the names of each of the viruses that were first included in the group, papilloma, polyoma, vacuolating virus (Melnick, 1962). The viruses are 40–57 nm in diameter and, as determined by negative staining, the outer shell has symmetry of the T = 7 icosahedral surface lattice and is composed of 72 morphological subunits (Finch and Klug, 1965; Anderer et al., 1967). The viruses contain no lipids and therefore are resistant to ether. Polyoma and SV40 do not share common antigens, nor is there evidence for the existence of any homology between their DNAs. The papovaviruses are capable of initiating a lytic cycle of replication or a latent infection. For the papilloma viruses it is difficult to obtain a suitable cell line in which the lytic cycle can be studied and for this reason studies that we will discuss concerning viral replication will deal exclusively with SV40 and polyoma.
Chapter
Viruses are the simplest biological structures possessing an autonomous genetic system, usually consisting of one macromole-cule of RNA or DNA, which controls the development and reproduction of the virion inside the host cell. In the simplest cases, virus RNA undergoes the same fate in the host’s cells as the messenger RNA which is synthesized in cell nuclei. For some period of time this virus RNA is fully adapted to conditions in the cell, using all the residual metabolic system of the cytoplasm or nucleus for synthesis of virus proteins. In some cases virus DNA becomes an element built into the host’s genetic system. Nevertheless, viruses can be regarded as autonomous genetic systems actively programming their development and reproduction.
Article
1. Electron microscopic observations were made on DNA fibers in the mitochondria of tumor cells induced by various kinds of DNA viruses, RNA viruses, and chemical carcinogens, and also in those of normal and regenerating rat livers and cultured liver cells. Intramitochondrial DNA fibers were observed most frequently in the sectioned specimens of adenovirus type 12-induced or SV 40-induced hamster tumor cells, but were hardly observed in normal and regenerating liver cells and other tumor cells. 2. The DNA fibers disappeared by treatment with DNase, but not with RNase. 3. The DNA fibers in adenovirus-induced tumor cells were easily isolated by osmotic shock and observed as circular DNA molecules by rotary shadowing with the electron microscope, while DNA molecules were hardly isolated from rat liver mitochondria by the same treatment. 4. On the ultracentrifugal fractionation of sonically disrupted adenovirus-induced tumor mitochondria, DNA were found to be contained mostly in the supernatant fraction by chemical analysis and by electron microscopic observation. 5. DNA fibers observed in the mitochondrial matrix in the sectioned specimens were proved to be identical with the isolated circular DNA molecules. 6. On the ultracentrifugal fractionation of sonically disrupted rat liver mitochondria, DNA were found to be contained mostly in the membrane fractions by chemical analysis. However, DNA molecules were difficult to observe with the electron microscope in the sectioned specimens of the rat liver mitochondrial membrane fractions. Nevertheless, they were isolated by phenol extraction from the membrane fractions, and were observed as circular DNA molecules with the electron microscope. 7. Appearance of the DNA fibers in the mitochondrial matrix seems to be associated with the division cycle of mitochondria, and in the mitochondria where DNA fibers are not observed in the sectioned specimens, the DNA molecules are supposed to be hidden by firmly attaching to the inner membrane, and to be isolated by chemical extraction.
Article
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Site-specific recombination is employed widely in bacteria and bacteriophage as a basis for genetic switching events that control phenotypic variation. It plays a vital role in the life cycles of phages and in the replication cycles of chromosomes and plasmids in bacteria. Site-specific recombinases drive these processes using very short segments of identical (or nearly identical) DNA sequences. In some cases, the efficiencies of the recombination reactions are modulated by the topological state of the participating DNA sequences and by the availability of accessory proteins that shape the DNA. These dependencies link the molecular machines that conduct the recombination reactions to the physiological state of the cell. This is because the topological state of bacterial DNA varies constantly during the growth cycle and so does the availability of the accessory factors. In addition, some accessory factors are under allosteric control by metabolic products or second messengers that report the physiological status of the cell. The interplay between DNA topology, accessory factors and site-specific recombination provides a powerful illustration of the connectedness and integration of molecular events in bacterial cells and in viruses that parasitise bacterial cells.
Article
We define the knotting probability of a knot $K$ by the probability for a random polygon (RP) or self-avoiding polygon (SAP) with $N$ segments having the knot type $K$. As a model of circular DNA we introduce the SAP consisting of impenetrable cylindrical segments of unit length in which the radius $r_{\rm ex}$ of segments corresponds to the screening length of DNA surrounded by counter ions. For various prime and composite knots we numerically show that a compact formula gives good fitted curves to the data of the knotting probabilities for the cylindrical SAP as a function of segment number $N$ and cylindrical radius $r_{\rm ex}$. It connects the small-$N$ to the large-$N$ regions and even to lattice knots for large values of radius $r_{\rm ex}$ such as satisfying $2 r_{\rm ex}= 1/4$. We suggest that if radius $r_{\rm ex}$ is large, the trefoil knot and its composite knots are dominant among the nontrivial knots in SAPs. We then study topological swelling that the mean-square radius of gyration of the cylindrical SAP with fixed knot is much larger than that of under no topological constraint if radius $r_{\rm ex}$ is small and $N$ is large enough. We argue that the finite-size effect is significant in it where the characteristic length of the knotting probability gives the topological scale. We show that for any value of radius $r_{\rm ex}$ a three-parameter formula gives a good fitted curve to the plot of the mean-square gyration radius of the cylindrical SAP with a given knot $K$ against segment number $N$. With the curves we evaluate the effective scaling exponent. We suggest that it increases with respect to the upper limit of $N$ and gradually approaches the scaling exponent of self-avoiding walks even in the case of zero thickness as the upper limit of $N$ becomes infinitely large.
Article
The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.
Preprint
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In this article, we present a black-hole-aided deep-helix (bh-dh) channel model to enhance information bound and mitigate a multiple-helix directional issue in Deoxyribonucleic acid (DNA) communications. The recent observations of DNA do not match with Shannon bound due to their multiple-helix directional issue. Hence, we propose a bh-dh channel model in this paper. The proposed bh-dh channel model follows a similar fashion of DNA and enriches the earlier DNA observations as well as achieving a composite like information bound. To do successfully the proposed bh-dh channel model, we first define a black-hole-aided Bernoulli-process and then consider a symmetric bh-dh channel model. After that, the geometric and graphical insight shows the resemblance of the proposed bh-dh channel model in DNA and Galaxy layout. In our exploration, the proposed bh-dh symmetric channel geometrically sketches a deep-pair-ellipse when a deep-pair information bit or digit is distributed in the proposed channel. Furthermore, the proposed channel graphically shapes as a beautiful circulant ring. The ring contains a central-hole, which looks like a central-black-hole of a Galaxy. The coordinates of the inner-ellipses denote a deep-double helix, and the coordinates of the outer-ellipses sketch a deep-parallel strand. Finally, the proposed bh-dh symmetric channel significantly outperforms the traditional binary-symmetric channel and is verified by computer simulations in terms of Shannon entropy and capacity bound. Keywords :A black hole, a black-hole-aided Bernoulli-process, DNA, a deep-helix, a deep-pair-ellipse, a beautiful-circulant-ring, a central-black-hole, a deep-double helix, and a deep-parallel strand, and a composite like information bound.
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In this article, we present a black-hole-aided deep-helix (bh-dh) channel model to enhance information bound and mitigate a multiple-helix directional issue in Deoxyribonucleic acid (DNA) communications. The recent observations of DNA do not match with Shannon bound due to their multiple-helix directional issue. Hence, we propose a bh-dh channel model in this paper. The proposed bh-dh channel model follows a similar fashion of DNA and enriches the earlier DNA observations as well as achieving a composite like information bound. To do successfully the proposed bh-dh channel model, we first define a black-hole-aided Bernoulli-process and then consider a symmetric bh-dh channel model. After that, the geometric and graphical insight shows the resemblance of the proposed bh-dh channel model in DNA and Galaxy layout. In our exploration, the proposed bh-dh symmetric channel geometrically sketches a deep-pair-ellipse when a deep-pair information bit or digit is distributed in the proposed channel. Furthermore, the proposed channel graphically shapes as a beautiful circulant ring. The ring contains a central-hole, which looks like a central-black-hole of a Galaxy. The coordinates of the inner-ellipses denote a deep-double helix, and the coordinates of the outer-ellipses sketch a deep-parallel strand. Finally, the proposed bh-dh symmetric channel significantly outperforms the traditional binary-symmetric channel and is verified by computer simulations in terms of Shannon entropy and capacity bound.
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Gene TherapyPlasmidsPlasmid BiopharmaceuticalsConclusions References
Article
The influence of distance dependent excluded volume interactions (EVIs) on the conformational properties of ring polymers is investigated within the principles of Rouse–Zimm theory. This study characterizes the structural features, i.e., the mean square radius of gyration, static structure factor, scattering intensity, fractal dimensions, and the mean square intermonomer distances of ring polymers in dilute solutions. The excluded volume interactions between the nonbonded monomers are modeled by a harmonic potential, where the strength of these interactions is evaluated from Flory’s mean-field approach. The mean square radius of gyration of the rings with EVI follows a similar scaling relation as its linear analog with the degree of polymerization. Unlike linear polymers, the maximum in the Kratky plot of the static structure factor is observed as a result of an increased segment density due to the circularity constraint. EVI marks a structural transition from relatively compact conformation for the smaller rings to expanded conformations for larger ones. The fractal dimension of the rings with EVI lies between those of the random walk and the self-avoiding walk models of linear polymers in three dimensions. This theoretical model produces a relatively pronounced effect of EVI for larger rings. An understanding of the influence of EVI on conformations of the ring polymers at a single molecule level may be applied to various naturally existing cyclic biomolecules such as plasmids, cyclic proteins, and polysaccharides.
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We study statistical properties of polymer chains with complex structures whose chemical connectivities are expressed by graphs. The multi-theta curve of m subchains with two branch points connected by them is one of the simplest graphs among those graphs having closed paths, i.e. loops. We denoted it by , and for m = 2 it is given by a ring. We derive analytically the pair distribution function and the scattering function for the θm-shaped polymer chains consisting of m Gaussian random walks of n steps. Surprisingly, it is shown rigorously that the mean-square radius of gyration for the Gaussian -shaped polymer chain does not depend on the number m of subchains if each subchain has the same fixed number of steps. For m = 3 we show the Kratky plot for the theta-shaped polymer chain consisting of hard cylindrical segments by the Monte-Carlo method including reflection at trivalent vertices.
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Bacterial genomes are now recognized as interacting intimately with cellular processes. Uncovering organizational mechanisms of bacterial genomes has been a primary focus of researchers to reveal the potential cellular activities. The advances in both experimental techniques and computational models provide a tremendous opportunity for understanding these mechanisms, and various studies have been proposed to explore the organization rules of bacterial genomes associated with functions recently. This review focuses mainly on the principles that shape the organization of bacterial genomes, both locally and globally. We first illustrate local structures as operons/transcription units for facilitating co-transcription and horizontal transfer of genes. We then clarify the constraints that globally shape bacterial genomes, such as metabolism, transcription and replication. Finally, we highlight challenges and opportunities to advance bacterial genomic studies and provide application perspectives of genome organization, including pathway hole assignment and genome assembly and understanding disease mechanisms.
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Modern bacteria regulate the expression of their genes through a spectrum of mechanisms ranging from the very simple to those that are highly complex. The regulatory mechanisms have evolved in concert with the means to detect changes to the physical or chemical environment, equipping the organism to respond to change. Regulation can be imposed at each stage of gene expression, and the networking of genes through coordinated control makes for multidimensional relationships that vary in time and space. Understanding how this regulatory complexity evolved is not a trivial matter, but it can be attempted. In one popular view, an ‘RNA world’ may have preceded the modern one with its DNA-based genomes. Looking for evidence of RNA-based gene regulation has been very fruitful and shows that gene control at this level is still very much in use: the conversion of genetic information held in RNA into protein by translation involves processes that are open to control at several levels. In DNA-based genomes, transcription is a fundamental process, and over evolutionary time bacterial cells have invested heavily in mechanisms that control it. Mechanisms that influence the activity of RNA polymerase are legion but fall into two categories: those that impede and those that assist the polymerase in the process of reading genetic information. It seems that simply turning genes on or off is rarely sufficient: it was necessary to evolve mechanisms for tuning transcription to the needs of the cell to promote survival, regardless of the size or level of sophistication of the organism’s genome. These regulatory mechanisms have evolved in ways that make their operations ‘noisy’, and this noise can be useful in generating physiological variety among genetically identical bacterial cells. It is becoming clear that the evolutionary forces that shape the bacterial nucleoid also guide the development of gene regulatory elements. For this reason the evolution of bacterial gene regulatory mechanisms will also be considered in the context of bacterial genome architecture.
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Our goal is to review the main theoretical models used to calculate free energy changes associated with common, torsion-induced conformational changes in DNA and provide the resulting equations hoping to facilitate quantitative analysis of both in vitro and in vivo studies. This review begins with a summary of work regarding the energy change of the negative supercoiling-induced B- to L-DNA transition, followed by a discussion of the energetics associated with the transition to Z-form DNA. Finally, it describes the energy changes associated with the formation of DNA curls and plectonemes, which can regulate DNA-protein interactions and promote cross talk between distant DNA elements, respectively. The salient formulas and parameters for each scenario are summarized in table format to facilitate comparison and provide a concise, user-friendly resource.
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L'expression des génomes dépend de toutes les « contraintes » qui s'appliquent à l'ADN, que ce soit au niveau nucléotidique et génique (1D), qu'au niveau de son organisation conformationnelle (3D). Les études menées chez les bactéries ont montré que l'organisation 3D de leur chromosome circulaire s'échelonne du niveau moléculaire au niveau cellulaire. Au niveau moléculaire, les NAPs (Nucleoid Associated Proteins) organisent l'ADN en microdomaines d'environ 10 kb qui présentent des surenroulements de l'hélice d'ADN indépendants (Postow et al., 2004). Au niveau cellulaire, le chromosome est organisé en 4 régions structurées et 2 autres non structurées, appelées macrodomaines, d'une taille proche de 1Mb (Valens et al., 2004). Le projet consiste à étudier l'impact de contraintes topologiques sur la transcription des gènes en fonction de la position chez E. coli. La taille élevée du chromosome et les éléments interagissant avec rendent variable et dynamique l'organisation chromosomique. Il est donc difficile d'étudier certains aspects de la structure du chromosome et de corréler ces résultats avec l'expression génique.
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The segmental relaxation dynamics of ring polymers in dilute solutions is investigated via optimized Rouse-Zimm theory. To the best of our knowledge, this is the first study that characterizes the orientational relaxation dynamics of ring polymers in dilute solutions. The orientational time autocorrelation functions are governed by two major processes that span a broad range of timescales: (i) local segmental motion at short times, independent of the ring size, (ii) overall motion of the ring at long times, that depends on the limiting ring size. Smaller rings relax faster than the larger rings and their respective linear analogues. The hydrodynamic interactions decrease the higher relaxation rates corresponding to the local relaxation modes and increase the smaller relaxation rates which correspond to the collective relaxation modes. The spectral density is independent of frequency in the low frequency regime while it decreases with increasing frequency. Regardless of the ring size, spin-lattice relaxation rate exhibits a single characteristic maximum as a function of frequency that shifts to a lower value with increasing strength of hydrodynamic interactions.
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The ATP-dependent DNase from Hemophilus influenzae has an unusual substrate specificity. The enzyme attacks linear duplex DNA to give initial products which are several hundred base pairs in length, suggesting an endonucleolytic mechanism. However, the enzyme differs from many other endonucleases in that it will not attack either single or double strand circular DNA, or duplex circles with nicks or gaps. These findings suggest that the enzyme requires a substrate with a free end. This requirement may be topological, as various enzymatic modifications of the termini do not affect the activity of the enzyme.
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We describe the properties of three DNA polymerases isolated from nuclear and cytoplasmic fractions of freshly harvested KB cells. Two of the enzymes, cytoplasmic polylymerase and nuclear polymerase N1, have been highly purified, one of them to apparent homogeneity. The third enzyme, nuclear polymerase N2, which is only partially purified, resembles the cytoplasmic enzyme in its physical properties but possesses distinctive enzymatic characteristics that suggest it may represent an independent third protein. The KB enzymes initiate polymerization at 3′-hydroxyl termini and resemble Escherichia coli DNA polymerase II in their obligatory requirement for gap-containing templates. None of the enzymes can copy the ribonucleotide strand of hybrid templates, even under specific conditions in which E. coli DNA polymerase I does demonstrate such activity. They can, however, with low efficiency copy a deoxynucleotide strand using an oligoribonucleotide as primer. Elucidation of the possible in vivo roles of these enzymes in DNA replication, recombination, and repair must await further biochemical and genetic studies.
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DNA conformation, in particular it's supercoiling, plays an important structural and functional role in gene accessibility as well as in DNA condensation. Enzyme driven changes of DNA plasmids between its linear, circular and supercoiled conformations control the level of condensation and DNA distal-site interactions. Many efforts have been made to quantify the branched supercoiled state of the DNA to understand its ubiquitous contribution to many biological functions, such as packaging, transcription, replication etc. Nanopore technology has proven to be an excellent label-free single-molecule method to investigate conformations of the translocating DNA in terms of the current pulse readout. In this paper, we present a comprehensive study to detect different branched-supercoils on individual plasmid DNA molecules. Using a detailed event charge deficit (ECD) analysis of the translocating molecules, we reveal, for the first time, the distributions in size and the position of the plectoneme branches on the supercoiled plasmid. Additionally, this analysis also gives an independent measure of the effective nanopore length. Finally, we use our nanopore platform for measurement of enzyme-dependent linearization of these branched-supercoiled plasmids. By simultaneous measurement of both single-molecule DNA supercoiled conformations as well as enzyme-dependent bulk conformational changes, we establish nanopore sensing as a promising platform for an in-depth understanding of structural landscapes of supercoiled DNA to decipher its functional role in different biological processes.
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Circular nucleic acids (CNAs) refer to nucleic acid molecules with a closed‐loop structure. This feature comes with a number of advantages, including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology controlled or enabled molecular devices. This article will begin with summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts on coupling functional nucleic acids with rolling circle amplification for ultra‐sensitive biosensing, and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery, will be recapitulated.
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Simple Summary: We comparatively discuss all possible methodologies for the complex DNA damage in situ detection. Fluorescent microscopy (FM) is the universal approach utilized in every technique, thus we discuss several FM variants. As image colocalization analysis is involved in the majority of methods, the related coefficients are reported. We envision this work to be a reference for radiobiologists in order to select and orchestrate the appropriate experimental and analysis strategies for optimized DNA damage detection. Last but not least, in the long term to help the incorporation of DNA damage biomarkers in the clinic as useful diagnostic or prognostic indicators. Abstract: Complexity of DNA damage is considered currently one if not the primary instigator of biological responses and determinant of short and long-term effects in organisms and their offspring. In this review, we focus on the detection of complex (clustered) DNA damage (CDD) induced for example by ionizing radiation (IR) and in some cases by high oxidative stress. We perform a short historical perspective in the field, emphasizing the microscopy-based techniques and methodologies for the detection of CDD at the cellular level. We extend this analysis on the pertaining methodology of surrogate protein markers of CDD (foci) colocalization and provide a unique synthesis of imaging parameters, software, and different types of microscopy used. Last but not least, we critically discuss the main advances and necessary future direction for the better detection of CDD, with important outcomes in biological and clinical setups.
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A tightly packed regularly coiled DNA ring is assumed to be present inside the polyoma virion. Applying the “one-step” technique of nucleic acid extraction from virions while being adsorbed to a protein monolayer, we studied the configurational changes of the DNA. Intact polyoma virions were mixed with DFP-trypsin (100μg/ml) and spread onto a subphase of a mixture of urea and sodium perchlorate (6 M and 2 M , or 3 M and 1 M respectively), at pH 8.2 and room temperature. After spreading the film, aliquots were transferred to grids at different periods of time, and the DNA stained by acetonic uranyl acetate. The length distribution of the DNA molecules measured from electron micrographs is shown in Fig. 1. The number of twists present at different times after spreading were also counted. At 12 minutes, with 3M urea and 1M sodium perchlorate in the subphase, the DNA was found to be 85% supertwisted (Fig. 2); 12% was opened to rings, and 3% existed as linear filaments.
Article
DNA buckling is the fundamental step for plectoneme nucleation and supercoil dynamics that are critical in the processing of genetic information. Here we systematically quantify DNA buckling dynamics using high-speed magnetic tweezers. Buckling times are ∼10–100ms and depend exponentially on both applied force and twist. By deconvolving measured time traces with the instrument response, we reconstruct full two-dimensional extension-twist energy landscapes of the buckling transition that reveal an asymmetry between the pre- and postbuckling states and suggest a highly bent transition state conformation.
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DNA extracted from purified polyoma virus contains closed and open molecules which are separable by velocity sedimentation either in the analytical or in the preparative ultracentrifuge. The closed configuration of the DNA gives rise to the fast band (component I) and the open configuration to a slow band, which is in some cases double (components II and III). Components I and II are able to produce plaques on mouse embryo monolayers, i.e., they possess cytocidal activity but which of the components cause cell transformation could not be determined owing to the lack of an adequate quantitative assay for transformation. A determination of the role of these DNA components in transformation is important, since it may clarify the mechanism of transformation. Such a determination is now made possible by the development of a sensitive quantitative assay for transformation caused by polyoma virus. This method is based on the ability of the transformed cells to form colonies in agar where the vast majority of the untransformed cells are unable to form colonies. Preliminary experiments have demonstrated that this method is also suitable for the study of the transforming ability of polyoma DNA.
Article
The enzymatic degradation of DNA has been followed by a continuous titration method and simultaneously by light scattering or viscosity. The resulting data, which are in terms of the number of bonds broken per parent molecule and the molecular weight decay, are compared with the expected molecular weight decay, calculated on the basis of several simple models. The results indicate that the molecule is composed of two polynucleotide strands and that the enzyme cleaves phosphorusester linkages at random. The maximum number of contiguous hydrogen bonds which fail to keep two fragments united is estimated at 2 pairs at 25°. Non-coincident ester-bond breaks have little effect on the over-all molecular configuration. The intact double helix appears to be "free-draining" in character, as evidenced by the simple proportionality between intrinsic viscosity and molecular weight; this is in contrast to the impermeability of free polynucleotide chains. Finally, it appears that a double helix in which there are numerous interruptions in the polynucleotide chains is incompatible with the evidence.
Article
A potentiometric technique for determination of protein-protein association constants is described. The technique consists of measuring the average number of protons (q̄) released when solutions of protein A and protein B (at the same pH) are mixed and thus allowed to form complex AB. Relations between q̄ and the apparent association constant, K app, were developed and employed. The association constants and q̄ values were determined for trypsin-soybean trypsin inhibitor system at 20.0° in 0.5 M KCl, 0.05 M CaCl 2 in the pH range 3.75-5.75 (and, by extrapolation of q̄, up to pH 8.30). The results are in satisfactory agreement with the few scattered data in the literature. An interesting kinetic phenomenon of "overshoot" in the soybean trypsin inhibitor-trypsin association was observed in this study. It was tentatively explained on the assumption that the association reaction is faster than mixing, whereas the dissociation reaction is measurably slow.
Article
High-titer polyma virus lysates were routinely obtained through the use of tissue cultures made from the kidneys of suckling mice that were inoculated with virus a few days after birth. The virus was then concentrated by sedimentation and purified by equilibrium centrifugation in a cesium chloride density gradient. By this procedure 316-fold and 375-fold increases of the ratio infectivity to protein content were obtained.Evidence in support of homogeneity of the purified virus preparations was obtained from analytical ultracentrifuge studies. Sedimentation velocity experiments revealed a single sedimenting component with a sedimentation constant, S20 w, of 242 ± 1 Svedbergs; equilibrium centrifugation in a cesium chloride density gradient showed the presence of a single symmetrical concentration distribution. Additional support for homogeneity was obtained from electron microscope observations and the ultraviolet absorption spectrum.After equilibrium centrifugation in a cesium chloride density gradient, crude polyoma virus fractionates into two main bands at levels corresponding to densities 1.339 g/ml and 1.297 g/ml. At density 1.339, virus displayed infectivity comparable to its hemagglutinating activity, showed an ultraviolet absorption spectrum typical of a nucleoprotein, and appeared to consist mainly of “full” particles in the electron microscope. At density 1.297, virus was characterized by hemagglutinating activity without corresponding infectivity, a typical protein-like ultraviolet absorption spectrum, and the appearance of “empty” particles in the electron microscope. Using the difference in buoyant densities between the two types of polyoma particles, the amount of deoxyribonucleic acid in the “full” particles was estimated as 13.4%.
Article
Analytical zone sedimentation (Vinograd, Bruner, Kent & Weigle, 1963) is shown to be a sensitive and reliable method for detecting differences in conformation and for determining the molecular weight of homogeneous phage DNA's. Changes in sedimentation coefficient as a function of pH and ionic strength reveal transitions between native and denatured DNA and between different forms of denatured DNA. In alkaline solution, denatured DNA appears to exist in a random coil conformation, whereas at neutral pH it is contracted by intrastrand base-base interactions to a degree determined by the ionic strength. The denatured forms appear to be the single strands of the native DNA double helix. Calibrations of sedimentation coefficient against molecular weight are derived for single -stranded DNA in neutral and alkaline molar sodium chloride and for native DNA in molar sodium chloride. Degradation of homogeneous DNA is sensitively detected, and endonuclease I of Escherichia coli is shown to make double-strand breaks in native DNA, as distinct from pancreatic DNase which makes single-strand breaks.
Article
A φX-DNA preparation cannot be inactivated, and can be hydrolysed only to a small extent, by E. coli phosphodiesterase. This result is a consequence of the unavailability of a free 3′-OH terminus. Studies of the action of spleen phosphodiesterase upon φX DNA similarly indicate the absence of a free 5′-0H terminus. Inactivation of φX. DNA by spleen or venom phosphodiesterase is exclusively a consequence of endonuclease activities present in these enzyme preparations. Pre-treatment of φX DNA with bacterial Phosphomonoesterase does not increase its susceptibility to the action of E. coli or spleen phosphodiesterase other than by the production of fragments as a consequence of contaminating endonuclease action.
Article
An automatic photoelectric scanning system is described for use with absorption optics in the ultracentrifuge. The system consists of a constant-speed drive assembly which by means of a flexible, self-centering coupling causes a lead screw to rotate, thereby imparting linear motion to the photomultiplier-slit assembly. In 5 sec. the photomultiplier scans a magnified image of the ultracentrifuge cell. High resolution is achieved by the use of a slit about 25 μ in width in front of the photocathode. Appropriate electronic circuits based upon well-established data sampling techniques provide for simultaneous measurement of the intensity of light and its derivative or, alternatively, the optical density and its derivative as a function of distance in the cell. Direct measurement of light intensity furnishes a sensitive index for the proper alignment of the optical system; optical density measurements supply the necessary data for sedimentation velocity and sedimentation equilibrium experiments. Detailed consideration is given to tests of the performance of the instrument. Sedimentation velocity experiments with bushy stunt virus show that results of high accuracy are obtained readily. These data include not only the determination of sedimentation coefficients but also the concentration and its change with time during an experiment. Results of molecular weight determinations of adenosine by sedimentation equilibrium are also given. The precision of these measurements is high. Although the accuracy also seems to be high, certain reservations are discussed and further work is outlined which should lead to greater reliability. At its present state of development the automatic scanner represents a vast improvement over the photographic method in terms of versatility, convenience, and precision. As yet the full potential of the method, especially with regard to accuracy, has not been realized.
Article
This communication presents a new method of carrying out sedimentation velocity experiments. A thin lamella of a solution of macromolecules is layered onto a denser miscible liquid in a rotating ultracentrifuge cell. The macromolecules then sediment through the liquid in a narrow concentration distribution, or band, which is observed photographically as a function of time. The density gradients necessary to stabilize the system against convection are generated during the experiment by the diffusion of small molecules between the lamella and the bulk solution, and in some cases by the sedimentation of the small molecules in the bulk solution. The inhomogeneities in the solvent are usually small enough to have no observable effect on the motion of the macromolecules. Sedimentation coefficients of macromolecules may be evaluated from the motion of bands. Diffusion, hydrodynamic interactions, and chemical reactions affect the shapes of bands.
Article
The isolation and the physical characterization of the DNA from T7 bacteriophage are described. The DNA is shown to be homogeneous by ultracentrifugation and chromatography. It is probable that this DNA preparation yields a mono-disperse solution of molecules of molecular weight 19 × 106.
Article
Crystals have been obtained from purified preparations of SE polyoma virus. Specific infectivity measurements of the mother liquor, wash fractions, and the dissolved crystals indicate the viral nature of the crystals.
Article
The DNA of polyoma (PY) virus has two interesting features: it is unusually resistant to heat or formamide denaturation,(1) and it has two distinct components, of sedimentation coefficient 14 and 21, respectively.(2) A two-component sedimentation was formerly shown to be a characteristic of the DNA of papilloma virus,(3) which is considered a member of the same group of viruses.
Article
When a preparation containing the DNA of the bacteriophage øX174 in its replicative form together with DNA from Escherichia coli is treated with exonucleases, the replicative form retains its characteristic physical properties, while the bacterial DNA is degraded. The evidence suggests that the mature replicative form exists as a closed ring.
Article
Electron micrographs of surface films containing the replicative form of the DNA of bacteriophage, øX174 show ring structures, whose average contour length is 1.64 micro, which have the characteristic appearance of double-stranded DNA throughout most of their length.
Article
The DNA extracted from polyoma virus exhibits certain properties which have not been reported for other viral base-paired DNA's. (1) The DNA renatures monomolecularly. The loss of helical configuration does not impair biological activity. Heating at 100° for 10-20 min followed by rapid cooling does not reduce the infective titer. Dulbecco has given evidence suggesting that a fraction of the polyoma DNA molecules are cyclic. (2) This form would in part account for the above properties.
Article
The DNA of Shope rabbit papilloma virus has been shown to exist in two forms with the same molecular weight but different sedimentation coefficients, 21 s and 28 s. It is suggested that the 28 s component has the form of a double-stranded ring and that the 21 s component is a linear molecule formed by opening the ring at some specific point.
Article
DNA extracted from SV40 virus has been shown to comprise two components with different sedimentation coefficients. The fast, 21.2 S, component appeared to be a circular molecule and the slow, 16.1 S, component the linear molecule produced by opening the ring. The molecular weight of the DNA, as estimated from band width in equilibrium density gradient, was found to be 2.7 × 106, as compared to 3.2 × 106 as estimated from the sedimentation coefficient of the slow component. The general properties of the DNA were similar to those of polyoma DNA except for the base composition. SV40 DNA was found to contain 41% guanine plus cytosine, as compared to 48% for polyoma DNA.
• W Murikami
Murikami, W., Science, 142, 56 (1963).
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Crawford, L. V., and P. H. Black, Virology, 24, 388 (1964). 4Weil, R., these PROCEEDINGS, 49, 480 (1963).
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25 Burton, A., and R. L. Sinsheimer, Science, 142, 962 (1963).
The estimate of 50 was obtained for the case of a 10% lowering of the weight average molecular weight
• C A Thomas
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• Wm
17 Thomas, C. A., Jr., J. Am. Chem. Soc., 78, 1861 (1956). The estimate of 50 was obtained for the case of a 10% lowering of the weight average molecular weight. 18 Studier, F. Wm., J. Mol. Biol., 11, 373 (1965).