DNA Circles Research Articles

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3′,5,5′-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

Minimal genomic variability in Merremia mosaic virus isolates endemic in Merremia spp and cultivated tomato in Puerto Rico.

Merremia mosaic virus (MerMV), a bipartite begomovirus, was identified for the first time as a pathogen of commercial tomato plantings. Infection of tomato by MerMV caused mild leaf curling and yellow foliar mosaic symptoms. Herein, the MerMV was identified in symptomatic Merremia quinquefolia and M. aegyptia (Convolvulaceae) plants exhibiting bright yellow or yellow-green foliar mosaic symptoms, respectively. The full-length begomoviral components were amplified from total DNA isolated from two wild species of Merremia and commercial tomato plants during 1991-1998. The DNA was subjected to rolling circle amplification, restriction digestion, and DNA sequencing. The resultant 19 and 26 apparently full-length DNA-A and DNA-B components were~2557 and~2492 bases, respectively. The 140-base common region was 97.9% identical between DNA-A and -B components, a predictive evidence for cognate DNA-A and -B components. Although the DNA-A components were highly conserved at 96-100%, the DNA-B components diverged at~89 to 100%, respectively. The overall clonal genomic features strongly suggested that MerMV lineage has been under host-selection for some time, and only recently, has undergone a host-shift, putatively, from wild convolvulaceous species to tomato (Solanaceae). Phylogenetically, MerMV grouped with other bipartite begomoviruses indigenous to the Caribbean region, with MerMV DNA-A components forming three clusters, and the DNA-B components grouped in one clade. Both clades contained only one closet relative, an isolate of MerMV from Venezuela, MerMV-VE. Biolistic inoculation of M. quinquefolia and tomato seedlings with the DNA-A and -B components of PR68 and PR80 resulted in development of symptoms like those observed in naturally-infected species, respectively.

Detection and characterization of tomato leaf curl New Delhi virus association with mosaic disease of ivy gourd (Coccinia grandis (L.) Voigt) in North India

Sixteen ivy gourd ( Coccinia grandis (L.) Voigt) plant samples showing severe mosaic symptoms were collected from New Delhi and Varanasi (Uttar Pradesh) in India. Begomovirus infection was confirmed by PCR using begomovirus-specific primers. Amplified PCR products (1.2 kb fragments) were cloned and the sequence was characterized. Based on sequence analysis, begomovirus associated with the majority of ivy gourd samples (16) was found to be a member of a bipartite begomovirus species, which is closely related to tomato leaf curl New Delhi virus (ToLCNDV). Therefore, two samples of ivy gourd, IVG1-ND and IVG2-Var, were selected for full-length genome (DNA-A and DNA-B-like sequence) amplification by the rolling circle DNA amplification (RCA) method. Sequence analysis performed using the Species Demarcation Tool (SDT) program revealed that they share 89.5-91.3% (IVG1-ND) and 93.4-96.8% (IVG2-Var) nucleotide (nt) identity with the DNA-A-like sequence of ToLCNDV isolated from cucurbits and chilli, respectively. The IVG1-ND and IVG2-Var isolates shared 90% nt identity among themselves, indicating that they are two different strains of ToLCNDV. Similarly, SDT analysis of the DNA-B-like sequence of IVG1-ND and IVG2-Var exhibited showed 82.7-93.3% nt identity with the DNA-B-like sequences of ToLCNDV infecting cucurbits. The recombination analysis of DNA-A and DNB-B-like sequences showed that the greater part of their genome most likely originated from previously reported begomoviruses that are known to infect chilli and cucurbits through recombination. https://doi.org/10.2298/ABS170616051V Received: June 16, 2017; Revised: November 16, 2017; Accepted: November 29, 2017; Published online: December 21, 2017 How to cite this article: Venkataravanappa V, Narasimha Reddy LRC, Saha S, Subbanna SK, Manem KR. Detection and characterization of tomato leaf curl New Delhi virus association with mosaic disease of ivy gourd ( Coccinia grandis (L.) Voigt) in North India. Arch Biol Sci. 2018;70(2):339-47.

Heat stress promotes longevity in budding yeast by relaxing the confinement of age-promoting factors in the mother cell.

Although individuals of many species inexorably age, a number of observations established that the rate of aging is modulated in response to a variety of mild stresses. Here, we investigated how heat stress promotes longevity in yeast. We show that upon growth at higher temperature, yeast cells relax the retention of DNA circles, which act as aging factors in the mother cell. The enhanced frequency at which circles redistribute to daughter cells was not due to changes of anaphase duration or nuclear shape but solely to the downregulation of the diffusion barrier in the nuclear envelope. This effect depended on the PKA and Tor1 pathways, downstream of stress-response kinase Pkc1. Inhibition of these responses restored barrier function and circle retention and abrogated the effect of heat stress on longevity. Our data indicate that redistribution of aging factors from aged cells to their progeny can be a mechanism for modulating longevity.

Rolling circle amplification shows a sinusoidal template length-dependent amplification bias.

Biophysical properties of DNA such as its longitudinal and torsional persistence length govern many processes and phenomena in biology, DNA nanotechnology and biotechnology. It has, for example, long been known that the circularization efficiency of short DNA fragments shows a periodic pattern where fragments with integer helical turns circularize much more efficiently than those with odd helical half turns due to stronger stacking of duplex ends. Small DNA circles can serve as templates for rolling circle amplification (RCA), which is a common and extremely robust amplification mechanism for nucleic acids. We discovered a strong template length-dependent amplification efficiency bias of RCA with the same periodicity as B-DNA. However, stacking cannot explain the mechanism behind this bias as the presence of the polymerase in the bifurcation fork inhibits base stacking of ends. Instead, coarse-grained molecular dynamics simulations imply that different amplification efficiencies come from a varying fraying probability of the last two downstream base pairs. We conclude that an increased strain-promoted fraying probability can increase the polymerization rate compared to a relaxed template.

A Horizontally Transferred Autonomous Helitron Became a Full Polydnavirus Segment in Cotesia vestalis.

Bracoviruses associate symbiotically with thousands of parasitoid wasp species in the family Braconidae, working as virulence gene vectors, and allowing the development of wasp larvae within hosts. These viruses are composed of multiple DNA circles that are packaged into infective particles, and injected together with wasp’s eggs during parasitization. One of the viral segments of Cotesia vestalis bracovirus contains a gene that has been previously described as a helicase of unknown origin. Here, we demonstrate that this gene is a Rep/Helicase from an intact Helitron transposable element that covers the viral segment almost entirely. We also provide evidence that this element underwent at least two horizontal transfers, which appear to have occurred consecutively: first from a Drosophila host ancestor to the genome of the parasitoid wasp C. vestalis and its bracovirus, and then from C. vestalis to a lepidopteran host (Bombyx mori). Our results reinforce the idea of parasitoid wasps as frequent agents of horizontal transfers in eukaryotes. Additionally, this Helitron-bracovirus segment is the first example of a transposable element that effectively became a whole viral circle.

Triplex DNA Nanostructures: From Basic Properties to Applications

Triplex nucleic acids have recently attracted interest as part of the rich "toolbox" of structures used to develop DNA-based nanostructures and materials. This Review addresses the use of DNA triplexes to assemble sensing platforms and molecular switches. Furthermore, the pH-induced, switchable assembly and dissociation of triplex-DNA-bridged nanostructures are presented. Specifically, the aggregation/deaggregation of nanoparticles, the reversible oligomerization of origami tiles and DNA circles, and the use of triplex DNA structures as functional units for the assembly of pH-responsive systems and materials are described. Examples include semiconductor-loaded DNA-stabilized microcapsules, DNA-functionalized dye-loaded metal-organic frameworks (MOFs), and the pH-induced release of the loads. Furthermore, the design of stimuli-responsive DNA-based hydrogels undergoing reversible pH-induced hydrogel-to-solution transitions using triplex nucleic acids is introduced, and the use of triplex DNA to assemble shape-memory hydrogels is discussed. An outlook for possible future applications of triplex nucleic acids is also provided.

Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

Highlights of This Issue

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective cancer therapy that uses an antibody-photoabsorber conjugate (APC) activated by NIR light. NIR-PIT causes minimal damage to non-target tissues and is effective with a variety of antibodies conjugated to the same photoabsorber. Prostate-specific membrane antigen (PSMA) is expressed in more aggressive prostate cancers and is an attractive target for NIR-PIT. Nagaya and colleagues report that NIR-PIT using anti-PSMA Ab-photoabsorber conjugate induced therapeutic effects against a PSMA-expressing prostate cancer in animal models. NIR-PIT could become a new treatment modality for primary and local-recurrent prostate cancer that could be readily translated to humans.Aberrant Notch signaling is critical in the initiation and maintenance of the neoplastic phenotype in many human neoplasms. It has long been appreciated that Notch functions as a transcriptional activator and establishes context dependent transcription cascades. What has been missing in the understanding of Notch-mediated transcriptional regulation is how Notch induces gene repression. Han and colleagues provide strong evidence that Notch directly recruits LSD1 and PRC2 to target promoters, which results in transcriptional repression. Not only does this study provide a novel paradigm of Notch signaling but also provides a rationale for developing new therapeutic strategies for Notch-dependent cancers.Prior work reported a large pool of small extrachromosomal circular DNA, microDNA, in normal tissues and cancer cell lines. Kumar and colleagues have now examined whether these circles of DNA, released from cells, can be detected in the blood. MicroDNA are found in the blood, and are released from cancers into the blood. Liquid biopsy is an experimental method being developed to detect cancers by examining the circulating cell-free linear DNA in the blood. These results suggest that circles of DNA are a new pool of circulating DNA that can be specifically examined in liquid biopsies.Diffuse intrinsic pontine glioma (DIPG) is an incurable childhood brain cancer whose hallmark mutation is H3K27M. Prior work unraveled that H3K27M inhibits polycomb repressive complex 2 (PRC2), an H3K27 methyltransferase complex. However, the genes downstream of H3K27M that contribute to tumorigenesis are not known. Here, using mouse genetics it is revealed that p16 (CDKN2A) is a target of H3K27M. The mechanism of p16 repression is unclear but involves aberrant histone and DNA methylation at the p16 promoter. Strikingly, the pro-tumorigenic effects of H3K27M were absent in mice with p16/p19 deletion, suggesting that restoring p16 levels is a promising therapeutic strategy.

Novel PCR primers to diagnose visceral leishmaniasis using peripheral blood, spleen or bone marrow aspirates.

To establish a suitable method of diagnosis of visceral leishmaniasis (VL) using peripheral blood, spleen or bone marrow aspirates. Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients (n=26). A new PCR primer pair (MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.

Detection of the alternative lengthening of telomeres pathway in malignant gliomas for improved molecular diagnosis.

Human malignant gliomas exhibit acquisition of either one of two telomere maintenance mechanisms, resulting from either reactivation of telomerase expression or activation of an alternative lengthening of telomeres (ALT) mechanism. In the present study, we analyzed 63 human malignant gliomas for the presence of ALT-specific extrachromosomal circles of telomeric DNA (C-circles) and measured telomerase expression, telomeric DNA content (Telo/Alu method), and telomeric repeat-containing RNAs (TERRA) levels. We also assessed histomolecular markers routinely used in clinical practice. The presence of C-circles significantly correlated with IDH1/2 mutation, MGMT exon 1 methylation, low Ki-67 immunostaining, increased telomeric DNA content, absence of functional ATRX protein and level of HTERT gene expression. In multivariate analysis, we observed a trend to a correlation between elevated TERRA levels and increased survival. Interestingly, the C-circles assay allowed to detect ALT activation in glioblastomas exhibiting wild-type IDH1/2 and ATRX expression. These results suggest that, after the correlations uncovered here have been confirmed on larger numbers of tumors, telomeric markers might be useful in improving diagnosis. They also point out to the utility of using the specific, sensitive and quantitative C-circle and Telo/Alu assays that can work with as few as 30ng of tumor DNA.

Molecular characterization of a novel bipartite begomovirus isolated from Lycianthes biflora in China.

A bipartite begomovirus isolate GD was isolated from Lycianthes biflora plants showing yellow mosaic symptoms in Nanxiong, Guangdong Province, China. The apparently full-length DNA-A and DNA-B viral components were cloned after enrichment of circular DNA by rolling circle amplification, restriction digestion, cloning, and DNA sequencing. The DNA-A component (2752nt, KT582302) shares highest (80.2%) nucleotide (nt) sequence identity with tomato leaf curl Sulawesi virus [Indonesia-Sulawesi-Langowan F101-2006] (ToLCSuV- [ID-Sul -LanF09-06], FJ237618), reported in Indonesia as causing yellow leaf curl disease of chilli pepper. The DNA-B component (2704nt, KT582303) shares highest (76.3%) nt sequence identity with pepper yellow leaf curl Indonesia virus-[Indonesia-tomato2-2005] (PepYLCIV-[ID-Tom2-05 AB213599) reported in Indonesia, and associated with yellow leaf curl disease in tomato. Based on the ICTV guidelines for begomoviral species demarcation, the virus is a new, previously undescribed bipartite begomovirus species for which the name "Lycianthes yellow mosaic virus" is proposed.

Binding, sliding, and function of cohesin during transcriptional activation

The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3' ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.

Novel DNA sensor system for highly sensitive and quantitative retrovirus detection using virus encoded integrase as a biomarker.

In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions happening within nanometer dimensions to directly detectable micrometer sized DNA products. The system utilizes the unique integration reaction of IN to generate a surface anchored nicked DNA circle that serves as a substrate for rolling circle amplification and allows for specific, quantitative and sensitive detection of purified recombinant IN or virus particles with a detection limit of less than 30 virus particles per μL of sample. Moreover, by modifying the nucleotide sequences of the utilized DNA it was possible to tailor the system to distinguish between the highly pathogenic lentivirus HIV and the gammaretrovirus murine leukemia virus present in a given sample. Infections with HIV remain a major threat to global health with more than 2 million new infections and 1 million deaths each year. The sensitive and specific detection of HIV particles based on IN activity holds promise for the development of a new type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment.

The analysis of proteins and small molecules based on sterically tunable nucleic acid hyperbranched rolling circle amplification

In this work, we succeeded in establishing a new method for proteins and small molecules analysis based on the small molecule-linked DNA and nucleic acid hyperbranched rolling circle amplification (HRCA). Small molecule linked DNA by chemical modification was used as a flexible tool to study protein-small molecule interactions. The HRCA reaction which would produce signal amplification was regulated by the steric effect depending on whether the target proteins were present. In the implement of the proposed strategy, streptavidin (SA)-biotin and anti-digoxin antibody (anti-Dig)-digoxin were chosen as two model partners. Experimental results showed that the quantitative detection of SA and anti-Dig was realized both with nanomolar detection limits. The small molecules biotin and digoxin were also detected at nanomolar levels in a wide range of 1nM~100µM and 1nM~10µM, respectively. Meanwhile, the results indicated that the method had a favorable specificity in analyzing proteins or small molecules. Thus, it may be expected to quantitatively analyze some protein markers and small molecular drugs in complex biological samples.

A balance between elongation and trimming regulates telomere stability in stem cells.

Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), however the mechanisms governing telomere length homeostasis in these cell types are unclear. Here, we report that telomere length is determined by the balance between telomere elongation mediated by telomerase and telomere trimming, controlled by the homologous recombination proteins XRCC3 and Nbs1 that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles), respectively. We found that reprogramming of differentiated cells induces T-circle and single stranded C-rich telomeric DNA accumulation, indicating the activation of telomere trimming pathways that compensate telomerase dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs, suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus, tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs.

2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns.The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected.Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened.Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented.Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rolling Circle Amplification with Chemically Modified Nucleoside Triphosphates.

Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility with isothermal amplification strategies. In this context, rolling circle amplification (RCA) is a wide-spread enzymatic replication method in which small single-stranded DNA (ssDNA) circles serve as templates in primer extension reactions yielding very long, ssDNA products. RCA is a pivotal tool for the generation of biosensor and diagnostic devices and is currently evaluated for its usefulness to create novel drug delivery systems. This unit describes the experimental procedures for the synthesis of modified RCA products using dN*TPs bearing chemical alterations at any possible location of the nucleosidic scaffold. Two ligation methods are presented for the generation of the DNA nanocircles that serve as templates for RCA, followed by a description of the RCA method itself and an assessment of the nuclease resistance of the ensuing products. © 2016 by John Wiley & Sons, Inc.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.