Nuclease Digestion Research Articles

Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment.

Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.

Abstract LB-048: Investigating mechanism of action of oligonucleotides homologous to the telomere overhang in melanoma

Abstract Telomeres have become a popular target for novel cancer therapeutics because activation of telomerase is associated with development of immortality in nearly 90% of all cancers. It has been shown that administration of an oligonucleotide homologous to the telomere overhang called T-oligo (5’-GTTAGGGTTAG-3’) induces potent DNA damage responses (DDRs) in several cancers, including melanoma, and causes minimal toxicity to normal cells. Our study focuses on T-oligo which has the guanine rich character of the telomere 3’ overhang. The folding and stabilization of G-quadruplexes (G4) which are G-rich structures at the end of telomeres may be involved in regulation of telomere length and cell cycle regulation. To date, T-oligo has not been shown to form G4, although evidence suggests it has a G4-mediated mechanism of action. In this study, we investigate T-oligo's ability to form G4 structures in vitro. We hypothesize the additional secondary structure of G4 configured T oligo may confer increased stability, resistance to nucleases in serum and, initiate DDRs. G4 formation is a potential target since G4 presence can cause telomere uncapping leading to DDRs. In our studies, G4 T-oligo was formed by heating various concentrations of T-oligo in KCl solutions to 90°C and incubating at 60°C for 24-36 hours. G4 T-oligo formation was verified using native PAGE and NMR spectroscopy before subjecting it to DNase1 degradation and analyzing with native PAGE. We also analyzed the uptake and anti-proliferative ability of G4 T-oligo using FACS analysis and MTT assay respectively. The change in expression of TRF2 and JNK activity was performed using western blot analysis since T-oligo may induce activation of SAPK/JNK pathway. We further investigated the interaction between T-oligo and TRF2 using immunofluorescence (IF) with FITC labeled T-oligo in MM-AN melanoma cells. Densitometric analysis after nuclease digestion demonstrated a 52.6% decrease in T-oligo and a 20% decrease in G4 T-oligo as well as a 96% increase of single stranded T-oligo within the G4 T-oligo sample after 90 minutes, indicating nuclease resistance of G4-T-oligo. FACS analysis revealed G4 T-oligo and T-oligo had similar cellular uptake but G4 T-oligo was shown to be less effective in inhibiting cell proliferation suggesting the reduced anti-proliferative activity of G4 T-oligo is not likely attributed to cellular uptake. Western blot results showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively after 24 hour treatment. JNK has previously been demonstrated to be activated by G4 stabilizing ligands, providing more evidence that T-oligo may induce telomere dysfunction and upregulation of JNK activity. IF showed 80% co-localization of T-oligo and TRF2 in the nucleus suggesting T oligo may induce dissociation of TRF2 from the telomere shelterin complex causing DDRs. In conclusion, formation of G4 species by T-oligo may induce DDRs and future studies may provide insights into its mechanism of action and suggests new avenues for research. Citation Format: Mark Frakes, Luke Wojdyla, Marko Ivancich, Perlina Fortinberry, Pooja Vinay, Priyanka Pandey, Gagan Chhabra, Neelu Puri. Investigating mechanism of action of oligonucleotides homologous to the telomere overhang in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-048.

Histone H1 Limits DNA Methylation in Neurospora crassa.

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

A Structural Bisulfite Assay to Identify DNA Cruciforms

In the half century since the discovery of the double-helix structure of DNA, it has become increasingly clear that DNA functionality is based on much more than its sequence in a double-helical structure. Further advances have highlighted the importance of additional aspects of DNA structure: its packaging in the higher order chromatin structure, positioning of nucleosomes along the DNA, and the occurrence of non-helical DNA structures. Of these, the latter has been problematic to prove empirically. Here, we describe a method that uses non-denaturing bisulfite sequencing on isolated Arabidopsis thaliana nuclei to determine the location of cytosines positioned outside the double helix as a result of non-B-form DNA structures. We couple this with computational methods and S1 nuclease digest to reliably identify stable, non-B-form, cruciform structures. This enables us to identify a palindrome in the promoter of FLOWERING LOCUS T that forms a stable non-B-form structure. The stronger conservation of the ability to form a non-helical secondary structure than of the sequence suggests that this structure is biologically relevant.

Fluorescence Methods for Probing G-Quadruplex Structure in Single- and Double-Stranded DNA.

Interest in exploring G-quadruplex (G4) structures in nucleic acids is growing as it becomes more widely recognized that these structures have many interesting biological roles and chemical properties. Probing the G4-forming potential of DNA with dimethyl sulfate, polymerase stop assays, or nuclease digestion are three commonly used techniques that usually employ radio-isotopic labels for visualization. However, as fluorescent labeling methods have grown in popularity and versatility, many laboratories have moved away from the routine use of radio-isotopic methods. We have adapted traditional procedures for structural analysis of G4-forming DNA sequences by using fluorescent labels and capillary electrophoresis and demonstrate their application to well-studied G4 structures, including c-MYC PU27 G4. The three fluorescent assays described here allow interrogation of G4 structures in double- and single-stranded DNA substrates, using either chemical or enzymatic cleavage. When combined, these techniques can provide valuable information for the investigation of G4 topology and structure, as well as visualizing any structural effects caused by interaction of quadruplexes with the complementary C-rich DNA strand.

Open chromatin reveals the functional maize genome

Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome.

MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.

Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.

Characterization of Extended-Spectrum β-Lactamase-Carrying Plasmids in Clinical Isolates ofKlebsiella pneumoniaefrom Taiwan

We analyzed the replicon types, sizes, and restriction fragment length polymorphism (RFLP) typing of plasmids carrying extended-spectrum β-lactamase (ESBL) genes in Klebsiella pneumoniae isolates from Taiwan. Fifty-one Escherichia coli transconjugant strains with plasmids from ESBL-producing K. pneumoniae from the Taiwan Surveillance of Antimicrobial Resistance III Program in 2002 were included. All the 51 plasmids carried a blaCTX-M gene, the majority of which were blaCTX-M-3 (28/51 [54.9%]). Plasmids ranged in size from 126 to 241 kb by S1 nuclease digestion and subsequent pulsed-field gel electrophoresis, and the most common plasmid size (37.3%) was 161-170 kb. The most common replicon type of plasmids was incompatibility group (Inc)A/C (60.8%). The IncA/C plasmids all carried blaCTX-M (blaCTX-M-3, -14, -15), and some also carried blaSHV (blaSHV-5, -12) genes. All 51 plasmids could be typed with PstI, and 27 (52.9%) belonged to 10 clusters. Thirty-eight of the 51 plasmids were typable with BamHI, and 21 plasmids (55.3%) fell into 7 clusters. Plasmids in the same cluster belonged to the same incompatibility group, with the exception of cluster C6. In conclusion, IncA/C plasmids are the main plasmid type responsible for the dissemination of ESBL genes of K. pneumoniae from Taiwan. RFLP with PstI possessed better discriminatory power than that with BamHI and PCR-based replicon typing for ESBL-carrying plasmids in K. pneumoniae in this study. Greater than 50% of plasmids fell into clusters, and >60% of cluster-classified plasmids were present in clonally unrelated isolates, indicating that horizontal transfer of plasmids plays an important role in the spread of ESBL genes.

Formation of RNA Beads from Various DNA Nanoring Structures.

By taking advantages of rolling circle transcription, one of the powerful methods to overcome the instability of RNA, we successfully synthesized RNA particles having high copy numbers of RNA strands. To examine dependence of RNA particle formation to template circular DNA strands, we synthesized RNA beads with 65-nt, 92-nt and 200-nt DNA nanoring structures as template circular DNA. Our conclusion is that characteristics of RNA beads could be controlled with various template circular DNA for RCT. It is feasible that our RNA beads could be used for RNAzyme-based metal sensors such as aqueous lead ion sensing. In this case, we expect that multi-metal ion detection would be possible by using 200-nt circular DNA bearing multiple desired functions as a template for RNA bead formation. Furthermore, certain features of RNA beads such as sensitivity to nuclease digestion and maximum loading amount of drugs when used as a carrier are expected to be further adjusted by choosing appropriate porosity and size.

Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors

Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5′ terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted.

Evaluating TNA stability under simulated physiological conditions

Chemically modified oligonucleotides are routinely used as diagnostic and therapeutic agents due to their enhanced biological stability relative to natural DNA and RNA. Here, we examine the biological stability of α-l-threofuranosyl nucleic acid (TNA), an artificial genetic polymer composed of repeating units of α-l-threofuranosyl sugars linked by 2′,3′-phosphodiester bonds. We show that TNA remains undigested after 7days of incubation in the presence of either 50% human serum or human liver microsomes and is stable against snake venom phosphordiesterase (a highly active 3′ exonuclease). We further show that TNA will protect internal DNA residues from nuclease digestion and shield complementary RNA strands from RNA degrading enzymes. Together, these results demonstrate that TNA is an RNA analogue with high biological stability.

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

Analysis of nuclease hypersensitive‐equivalent sites in SV40 by Next Generation Sequencing

Chromatin fragments obtained by sonication or Micrococcal nuclease digestion of SV40 minichromosomes or disrupted virions were analyzed by Next Generation Sequencing (NGS) to determine the location of regions of the SV40 genome differentially sensitive to either treatment. Both treatments yielded samples which upon analysis allowed us to identify regions of hypersensitivity and moderate sensitivity. With sonication all of the samples contained regions of hypersensitivity centered on nucleotides 141 and 221 and regions of moderate sensitivity surrounding these sites which included the 21 base pair repeat regions and enhancers. Following Micrococcal nuclease treatment Hypersensitive sites were found in minichromosomes centered on nucleotides 30 and 92 and in chromatin from disrupted virions centered at nucleotide 106. Micrococcal nuclease also generated a pattern of peaks and valleys based upon the number of reads at each nucleotide consistent with the location of nucleosomes. While the pattern was very similar throughout most of the SV40 genome in minichromosomes and chromatin from disrupted virions, the pattern differed significantly in the regulatory region. In minichromosomes we observed a nucleosome‐free region extending from approximately nucleotide 5000 to nucleotide 250 encompassing most of the early and late transcription regulatory sequences and the origin of replication. In chromatin from disrupted virions we observed a very small open region which at most included only the enhancers.Support or Funding InformationThis work was supported by grants from NIH AI094441 (to BM) and NIH GM098328 (to LB)

The high mobility group protein HMO1 functions as a linker histone in yeast.

BackgroundEukaryotic chromatin consists of nucleosome core particles connected by linker DNA of variable length. Histone H1 associates with the linker DNA to stabilize the higher-order chromatin structure and to modulate the ability of regulatory factors to access their nucleosomal targets. In Saccharomyces cerevisiae, the protein with greatest sequence similarity to H1 is Hho1p. However, during vegetative growth, hho1∆ cells do not show any discernible cell growth defects or the changes in bulk chromatin structure that are characteristic of chromatin from multicellular eukaryotes in which H1 is depleted. In contrast, the yeast high mobility group (HMGB) protein HMO1 has been reported to compact chromatin, as evidenced by increased nuclease sensitivity in hmo1∆ cells. HMO1 has an unusual domain architecture compared to vertebrate HMGB proteins in that the HMG domains are followed by a lysine-rich extension instead of an acidic domain. We address here the hypothesis that HMO1 serves the role of H1 in terms of chromatin compaction and that this function requires the lysine-rich extension.ResultsWe show here that HMO1 fulfills this function of a linker histone. For histone H1, chromatin compaction requires its basic C-terminal domain, and we find that the same pertains to HMO1, as deletion of its C-terminal lysine-rich extension renders chromatin nuclease sensitive. On rDNA, deletion of both HMO1 and Hho1p is required for significantly increased nuclease sensitivity. Expression of human histone H1 completely reverses the nuclease sensitivity characteristic of chromatin isolated from hmo1∆ cells. While chromatin remodeling events associated with repair of DNA double-strand breaks occur faster in the more dynamic chromatin environment created by the hmo1 deletion, expression of human histone H1 results in chromatin remodeling and double-strand break repair similar to that observed in wild-type cells.ConclusionOur data suggest that S. cerevisiae HMO1 protects linker DNA from nuclease digestion, a property also characteristic of mammalian linker histone H1. Notably, association with HMO1 creates a less dynamic chromatin environment that depends on its lysine-rich domain. That HMO1 has linker histone function has implications for investigations of chromatin structure and function as well as for evolution of proteins with roles in chromatin compaction.

Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

BackgroundThe nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed. MethodsProtocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries. ResultsWe present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms. ConclusionsOur practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.

Near Infrared-Guided Smart Nanocarriers for MicroRNA-Controlled Release of Doxorubicin/siRNA with Intracellular ATP as Fuel.

In chemotherapy, it is a great challenge to recruit endogenous stimuli instead of external intervention for targeted delivery and controlled release; microRNAs are the most promising candidates due to their vital role during tumorigenesis and significant expression difference. Herein, to amplify the low abundant microRNAs in live cells, we designed a stimuli-responsive DNA Y-motif for codelivery of siRNA and Dox, in which the cargo release was achieved via enzyme-free cascade amplification with endogenous microRNA as trigger and ATP (or H(+)) as fuel through toehold-mediated strand displacement. Furthermore, to realize controlled release in tumor cells, smart nanocarriers were constructed with stimuli-responsive Y-motifs, gold nanorods, and temperature-sensitive polymers, whose surfaces could be reversibly switched between PEG and RGD states via photothermal conversion. The PEG corona kept the nanocarriers stealth during blood circulation to protect the Y-motifs against nuclease digestion and enhance passive accumulation, whereas the exposed RGD shell under near-infrared (NIR) irradiation at tumor sites facilitated the specific receptor-mediated endocytosis by tumor cells. Through modulating NIR laser, microRNA, or ATP expressions, the therapy efficacies to five different cell lines were finely controlled, presenting NIR-guided accumulation, massive release, efficient gene silence, and severe apoptosis in HeLa cells; in vivo study showed that a low dosage of nanocarriers synergistically inhibited the tumor growth by silencing gene expression and inducing cell apoptosis under mild NIR irradiation, though they only brought minimum damage to normal organs. The combination of nanomaterials, polymers, and DNA nanomachines provided a promising tool for designing smart nanodevices for disease therapy.

CJD and Scrapie Require Agent-Associated Nucleic Acids for Infection.

Unlike Alzheimer's and most other neurodegenerative diseases, Transmissible Spongiform Encephalopathies (TSEs) are all caused by actively replicating infectious particles of viral size and density. Different strain-specific TSE agents cause CJD, kuru, scrapie and BSE, and all behave as latent viruses that evade adaptive immune responses and can persist for years in lymphoreticular tissues. A foreign viral structure with a nucleic acid genome best explains these TSE strains and their endemic and epidemic spread in susceptible species. Nevertheless, it is widely believed that host prion protein (PrP), without any genetic material, encodes all these strains. We developed rapid infectivity assays that allowed us to reproducibly isolate infectious particles where >85% of the starting titer separated from the majority of host components, including PrP. Remarkably, digestion of all forms of PrP did not reduce brain particle titers. To ask if TSE agents, as other viruses, require nucleic acids, we exposed high titer FU-CJD and 22L scrapie particles to potent nucleases. Both agent-strains were propagated in GT1 neuronal cells to avoid interference by complex degenerative brain changes that can impede nuclease digestions. After exposure to nucleases that are active in sarkosyl, infectivity of both agents was reproducibly reduced by ≥99%. No gold-stained host proteins or any form of PrP were visibly altered by these nucleases. In contrast, co-purifying protected mitochondrial DNA and circular SPHINX DNAs were destroyed. These findings demonstrate that TSE agents require protected genetic material to infect their hosts, and should reopen investigation of essential agent nucleic acids. J. Cell. Biochem. 117: 1947-1958, 2016. © 2016 Wiley Periodicals, Inc.

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

Nucleotide Catabolism on the Surface of Aortic Valve Xenografts; Effects of Different Decellularization Strategies

Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.

A one-dimensional statistical mechanics model for nucleosome positioning on genomic DNA

The first level of folding of DNA in eukaryotes is provided by the so-called ‘10 nm chromatin fibre’, where DNA wraps around histone proteins (∼10 nm in size) to form nucleosomes, which go on to create a zig-zagging bead-on-a-string structure. In this work we present a one-dimensional statistical mechanics model to study nucleosome positioning within one such 10 nm fibre. We focus on the case of genomic sheep DNA, and we start from effective potentials valid at infinite dilution and determined from high-resolution in vitro salt dialysis experiments. We study positioning within a polynucleosome chain, and compare the results for genomic DNA to that obtained in the simplest case of homogeneous DNA, where the problem can be mapped to a Tonks gas []. First, we consider the simple, analytically solvable, case where nucleosomes are assumed to be point-like. Then, we perform numerical simulations to gauge the effect of their finite size on the nucleosomal distribution probabilities. Finally we compare nucleosome distributions and simulated nuclease digestion patterns for the two cases (homogeneous and sheep DNA), thereby providing testable predictions of the effect of sequence on experimentally observable quantities in experiments on polynucleosome chromatin fibres reconstituted in vitro.