Linear DNA Sequence Research Articles

Heterogeneity of the Nuclear Environment Investigated by Superresolution Microscopy and Fluorescence Correlation Spectroscopy

Genomes are more than one-dimensional entities purely defined by their linear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge in biology is to decipher the principles of organization of what can be considered, by analogy with human-created libraries, the cell's primary unit of information storage and retrieval. In this respect, the development of super-resolved fluorescence microscopy has provided a new toolbox to peer into the nucleus. For instance, super-resolution microscopy has been recently applied to the investigation of nanoscale chromatin architecture, revealing nucleosome higher-order organization into heterogeneous ‘clutches’ and epigenetically dependent folding motives [Ricci M et al, Cell, (2015); Boettiger A et al, Nature (2016)].However, dynamic properties of proteins in the nucleus are also critical for their function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy (FCS) has been used to map the dynamics of several proteins in the nucleus with diffraction-limited spatial resolution. Here we combine a novel STED-based super-resolution method [Lanzano’ L et al, Nat Commun (2015)] with FCS to measure protein dynamics in the nucleus with an improved spatial resolution of about 100 nm. We sample several positions within the nucleus by performing line scanning. The measured spatial and temporal heterogeneity of the dynamics, quantified by a recently introduced algorithm [Scipioni L et al, Biophys J (2016)], is discussed in relation to nuclear organization.

Interference between Triplex and Protein Binding to Distal Sites on Supercoiled DNA

We have explored the interdependence of the binding of a DNA triplex and a repressor protein to distal recognition sites on supercoiled DNA minicircles using MD simulations. We observe that the interaction between the two ligands through their influence on their DNA template is determined by a subtle interplay of DNA mechanics and electrostatics, that the changes in flexibility induced by ligand binding play an important role and that supercoiling can instigate additional ligand-DNA contacts that would not be possible in simple linear DNA sequences.

CRISPR/Cas9 genome editing throws descriptive 3-D genome folding studies for a loop.

CRISPR/Cas9 genome editing studies have recently shed new light into the causal link between the linear DNA sequence and 3-D chromatin architecture. Here we describe current models for the folding of genomes into a nested hierarchy of higher-order structures and discuss new insights into the organizing principles governing genome folding at each length scale. WIREs Syst Biol Med 2016, 8:286-299. doi: 10.1002/wsbm.1338 For further resources related to this article, please visit the WIREs website.

Functional proteomic biomarkers in cancer.

Beyond penetrant germline and somatic mutations, there are substantial challenges in extrapolating phenotypes from linear DNA sequences and transcriptomics. This brings a molecular pathology emphasis to the properties of the main players responsible for executing actions, proteins. The proteomic attribute most frequently determined in pathology is (relative) content, but for many candidate biomarkers this is not the most important feature to understand. In keeping pace with the depth of knowledge of the mechanisms underlying pathologies, we need to ask more sophisticated questions about the state of proteins, for example, their oligomerization status, modification status, and location. This demands hitherto nonroutine approaches to proteomics, which we will discuss in this brief perspective.

Illuminating the genome

It was long assumed that our linear DNA sequence encodes all the information required to influence the development of multicellular organisms and their progeny. This notion is not surprising given the dramatic changes that occur in organisms when minor changes are made in the genetic code. However, the past few decades have challenged this simple concept, igniting a renaissance in nuclear cell biology.

3D Genome Architecture in the Mammalian Nucleus

Advances in DNA-sequencing technologies have made it possible to study the spatial hierarchy of genomic organization across different eukaryotic systems. Although genetic information is encrypted in the linear DNA sequence, a growing body of research suggests that the 3D genome plays an essential role in gene regulation [1]. The latest technological innovations associated with chromosome conformation capture methodologies (e.g., 3C, 4C, 5C, Hi-C and ChIA-PET) in mapping higher-order genomic communications have established new principles that describe spatial chromatin organization and its role in transcriptional control [2,3]. Experimental evidence indicates that the nuclear architecture is composed of a hierarchy of genomic interactions, from chromatin loops that connect genes and enhancers to larger chromosome territories and nuclear compartments [1,2,4].

Molecular Characterization of Herpes Simplex Virus 2 Strains by Analysis of Microsatellite Polymorphism

The complete 154-kbp linear double-stranded genomic DNA sequence of herpes simplex virus 2 (HSV-2), consisting of two extended regions of unique sequences bounded by a pair of inverted repeat elements, was published in 1998 and since then has been widely employed in a wide range of studies. Throughout the HSV-2 genome are scattered 150 microsatellites (also referred to as short tandem repeats) of 1- to 6-nucleotide motifs, mainly distributed in noncoding regions. Microsatellites are considered reliable markers for genetic mapping to differentiate herpesvirus strains, as shown for cytomegalovirus and HSV-1. The aim of this work was to characterize 12 polymorphic microsatellites within the HSV-2 genome by use of 3 multiplex PCR assays in combination with length polymorphism analysis for the rapid genetic differentiation of 56 HSV-2 clinical isolates and 2 HSV-2 laboratory strains (gHSV-2 and MS). This new system was applied to a specific new HSV-2 variant recently identified in HIV-1-infected patients originating from West Africa. Our results confirm that microsatellite polymorphism analysis is an accurate tool for studying the epidemiology of HSV-2 infections.

104 Probing DNA shape on molecular and genomic scales

Linear DNA sequence encodes its three-dimensional structure. The resulting DNA shape gives DNA unique physical and chemical properties that are explored by DNA-binding proteins (Rohs et al., 2009). Probing DNA shape, therefore, helps to reveal the origin of specificity in DNA-protein recognition. We have developed a Monte Carlo (MC) method for all-atom DNA structure prediction (Joshi et al., 2007). With key components such as parallelization and functionality to handle chemical modifications added, the revamped MC method can efficiently recover the DNA shape of any DNA sequence. To embrace the genomic era, we have trained a high-throughput (HT) prediction method on 2121 independent MC simulations (Slattery et al., 2011). Applying this method on whole genome DNA sequences of 66 Drosophila individuals, we found that among single nucleotide polymorphisms (SNPs) within the immediate vicinity of in vivo transcription factor (TF) binding sites, low-frequency SNPs tend to change the DNA minor groove width more than high-frequency SNPs. This indicates that shape-changing mutations are detrimental to TF binding and in vivo function. We also examined the dependency of DNase I cleavage events on DNA shape using our HT method. Our results show that the rate of DNase I cleavage closely tracks the width of the minor groove. MC predictions for chemically modified bases further demonstrate that cytosine methylation enhances cleavage directly adjacent to CpG dinucleotides through narrowing of the minor groove. In summary, with tools on hand to address biological questions on both molecular and genomic scales, our work provides new insights into gene regulation and evolution.

Complete Genome Sequence of Bp7, an Escherichia coli Bacteriophage with a Wide Host Range

Chicken colibacillosis is caused by some pathogenic Escherichia coli strains. Thirty-five pathogenic antibiotic-resistant E. coli strains were used in the host range detection of bacteriophage Bp7. The phage showed a wide range of E. coli hosts (46%). The complete genome of bacteriophage Bp7 was sequenced, assembled, and analyzed. The results revealed a linear double-stranded DNA sequence of 168,066 bp harboring 791 open reading frames. The major findings from its annotation are described.

Complete Genome Sequence of Salmonella Bacteriophage SS3e

A Salmonella lytic bacteriophage, SS3e, was isolated, and its genome was sequenced completely. This phage is able to lyse not only various Salmonella serovars but also Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens, indicating a broad host specificity. Genomic sequence analysis of SS3e revealed a linear double-stranded DNA sequence of 40,793 bp harboring 58 open reading frames, which is highly similar to Salmonella phages SETP13 and MB78.

Separation principles of cycling temperature capillary electrophoresis

High throughput means to detect and quantify low-frequency mutations (<10(-2) ) in the DNA-coding sequences of human tissues and pathological lesions are required to discover the kinds, numbers, and rates of genetic mutations that (i) confer inherited risk for disease or (ii) arise in somatic tissues as events required for clonal diseases such as cancers and atherosclerotic plaque.While throughput of linear DNA sequencing methods has increased dramatically, such methods are limited by high error rates (>10(-3) ) rendering them unsuitable for the detection of low-frequency risk-conferring mutations among the many neutral mutations carried in the general population or formed in tissue growth and development. In contrast, constant denaturing capillary electrophoresis (CDCE), coupled with high-fidelity PCR, achieved a point mutation detection limit of <10(-5) in exon-sized sequences from human tissue or pooled blood samples. However, increasing CDCE throughput proved difficult due to the need for precise temperature control and the time-consuming optimization steps for each DNA sequence probed. Both of these problems have been solved by the method of cycling temperature capillary electrophoresis (CTCE). The data presented here provide a deeper understanding of the separation principles involved in CTCE and address several elements of a previously presented two-state transport model.

SSDNA cutter v0.0 a new &amp;lt;i&amp;gt;in silico&amp;lt;/i&amp;gt; RFLP tool in C

Summary: SSDNA Cutter v0.0 is a new in silico RFLP tool written in C. It has a significant utility in studying population diversity as it has the capability of restriction digestion of a group of linear DNA se-quences and then segregate the restriction patterns into separate groups as per similarity in the restriction maps. The software has an inbuilt database for 20 restriction enzymes and it is flexible so that the user can add up to 100 more restriction enzymes to the list. The interface is easy, simple and interactive which enables the user to obtain restriction pattern groups which are quite similar to Operational Taxonomic Units obtained from phylogenetic trees. Availability: The software and the source code is currently available from the authors on request without any cost. We intend to release it in a public domain soon. SSDNA Cutter v0.0 is licensed under the GNU General Public License.

Polymorphism in exon 3 of follicle stimulating hormone beta ( FSHB) subunit gene and its association with litter traits and superovulation in the goat

The polymorphism in follicle stimulating hormone beta ( FSHB) subunit gene was detected by PCR-SSCP and PCR-RFLP methods in 780 does from four goat breeds including Boer, Matou, Black and Boer–Matou crossbred (BM). The associations of FSHB genotypes with litter size (LS), litter weight at birth (LWB) and gestation length (GL) as well as superovulation performances were analyzed in relatively lower-prolific Boer and higher-prolific Matou breeds. A 1514 bp linear DNA sequence of goat FSHB gene covering the complete 3 exons was cloned by four pairs of primer. A new mutation (A2645G, GenBank Accession no: S64745) locating in exon 3 causing an amino acid change from glutamine (Gln) to arginine (Arg) at residue 115 was identified, which resulted in three genotypes named AA, AB and BB. The three genotypes were detected in the four studied goat breeds. The higher-prolific Matou breed had the highest frequency of genotype AA. Association analysis showed that Boer and Matou does with AA genotype have the largest LS both in average and in parities from first to fourth ( P < 0.05). Furthermore, Boer does with AA genotype had the heaviest LWB compared to other two genotypes ( P < 0.05). Matou does with AA genotype have more numbers of ova harvested, large follicles and corpus lutea on ovaries after superovulation than those with BB genotype ( P < 0.05). Therefore, these results suggest that the FSHB gene is a candidate gene that affects reproduction traits in goats.

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

A simple method encoding linear single strain DNA sequence with natural numbers

A simple method presenting linear single strain DNA (LssDNA) sequence with natural numbers is introduced in this paper. The method presents LssDNA correspondingly with the numerals 1, 2, 3 and 4. After calculation, the sequence can be coded in natural numbers which can also be decoded into the DNA sequence. Thus, an LssDNA sequence can be expressed in a natural number and a dot at coordinate axes. In the future, a new LssDNA sequences database termed “DotBank” would be realized in which each LssDNA sequence is determined as a dot.

Comparative analysis of long DNA sequences by per element information content using different contexts

BackgroundFeatures of a DNA sequence can be found by compressing the sequence under a suitable model; good compression implies low information content. Good DNA compression models consider repetition, differences between repeats, and base distributions. From a linear DNA sequence, a compression model can produce a linear information sequence. Linear space complexity is important when exploring long DNA sequences of the order of millions of bases. Compressing a sequence in isolation will include information on self-repetition. Whereas compressing a sequence Y in the context of another X can find what new information X gives about Y. This paper presents a methodology for performing comparative analysis to find features exposed by such models.ResultsWe apply such a model to find features across chromosomes of Cyanidioschyzon merolae. We present a tool that provides useful linear transformations to investigate and save new sequences. Various examples illustrate the methodology, finding features for sequences alone and in different contexts. We also show how to highlight all sets of self-repetition features, in this case within Plasmodium falciparum chromosome 2.ConclusionThe methodology finds features that are significant and that biologists confirm. The exploration of long information sequences in linear time and space is fast and the saved results are self documenting.

Adsorption of DNA into Mesoporous Silica

In these experiments, double-stranded, linear DNA sequences were adsorbed into the pores of spherically shaped acid-prepared mesoporous silica (APMS). The lengths of the sequences were either 760 base pairs or 2000 base pairs. DNA adsorption into the interior of the mesoporous material was confirmed using confocal microscopy of sequences containing fluorescently labeled DNA molecules. Additional characterization with N(2) physisorption and powder X-ray diffraction supported this finding. The extent of adsorption was measured at various concentrations using UV-visible spectrophotometry to establish adsorption isotherms. APMS alone adsorbed a negligible amount of DNA; however, exchanging divalent cations such as Mg(2+) and Ca(2+) into the pores of APMS prior to DNA uptake was found to cause a significant amount of DNA to be adsorbed. Using Na(+) caused a lower amount of DNA to be adsorbed. DNA adsorption was also dependent on the pore diameter of APMS. Adsorption increased upon expansion of the pore size of the metal ion-exchanged material from 34 to 54 A; however, no additional uptake was measured by further increasing the pore size to 100 A. The amount of DNA adsorbed could also be significantly increased by using (aminopropyl)triethoxysilane to covalently link ammonium ions to the surface. Postsynthetic modification of the silica surface with aminopropyl groups increased the maximum DNA adsorption to 15.7 microg/mg silica, for materials with pore diameters of 100 A, which is 2 to 3 times more adsorbed DNA than for metal ion-exchanged material. This indicated that DNA binds more strongly in the presence of the ammonium group compared to the metal counterions. Finally, calculation and comparison of Freundlich and Langmuir constants for these adsorption processes indicate that intermolecular interactions between the DNA molecules within the pores are significant when the effective pore diameter is small, including materials with larger pores that were modified with organosilane.

Gene Order and Dynamic Domains

When considering the daunting complexity of eukaryotic genomes, some comfort can be found in the fact that the human genome may contain only 30,000 to 40,000 genes. Moreover, growing evidence suggests that genomes may be organized in such a way as to take advantage of space. A gene's location in the linear DNA sequence and its position in the three-dimensional nucleus can both be important in its regulation. Contrary to prevailing notions in this postgenomic era, the bacteriophage lambda, a paragon of simplicity, may still have a few things to teach us with respect to these facets of nonrandom genomes.

Reconstructing tumor amplisomes.

Duplication of genomic sequences is a common phenomenon in tumor cells. While many duplications associated with tumors have been identified (e.g. via techniques such as CGH), both the organization of the duplicated sequences and the process that leads to these duplications are less clear. One mechanism that has been observed to lead to duplication is the extraction of DNA from the chromosomes and aggregation of this DNA into small, independently replicating linear or circular DNA sequences (amplisomes). Parts of these amplisomes may later be reinserted back into the main chromosomes leading to duplication. Although amplisomes are known to play an important role in tumorigenesis, their architecture and even size remain largely unknown. We reconstruct the structure of tumor amplisomes by analyzing duplications in the tumor genome. Our approach relies on recently generated data from End Sequence Profiling (ESP) experiments, which allow us to examine the fine structure of duplications in a tumor on a genome-wide scale. Using ESP data, we formulate the Amplisome Reconstruction Problem, describe an algorithm for its solution, and derive a putative architecture of a tumor amplisome that is the source for duplicated material in the MCF7 breast tumor cell line.

Introduction

This is an auspicious time for plant cell biology. We have the recent genome sequence of the model plant Arabidopsis, together with powerful new sets of tools that include functional genomics and the dynamic imaging of green fluorescent protein (GFP)–conjugated proteins by confocal microscopy. We are well placed indeed for uncovering the ways in which all genes act out their roles within individual cells. It is the combinatorial properties of these individual cells that underpin both the higherorder processes of plant growth and development and of plant evolution. At a crucial level in the hierarchy of explanations and structures, between the linear DNA sequence at one extreme and whole plant phenomena at the other, lies the cell. It is to this cellular level, to the irreducible unit of life, that this collection of papers is addressed, literally ‘between genome and plant’. Most of the major conceptual advances in our understanding of cell biology have come from work with animal and fungal systems. The ways in which membranes create dynamic compartments within cells and the role of the cytoskeleton in providing an ordering framework are familiar from the textbooks. But plant cells have special features.